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Questions and Answers

What does the height of the rocket in an assay indicate?

Antibody purity

Rate of chemical reaction

Antigen concentration (correct)

Electrophoresis speed

What is a starting point for conducting a Latex Agglutination Assay?

Calculating optimal temperature

Preparing the standard and samples (correct)

Inoculating a culture

Diluting the antisera

In crossed immunoelectrophoresis, what is the second dimension of separation?

Ultrafiltration

Electrophoresis at right angles (correct)

Western blotting

Centrifugation

What can lead to the Hook effect in immunoassays?

High analyte concentration Signup and view all the answers

What is the role of antiserum in immunodiffusion assays?

To bind with proteins during precipitation Signup and view all the answers

Which is a characteristic of the Latex Agglutination Assay?

Can use various biological samples Signup and view all the answers

What does a precipitate indicate in crossed immunoelectrophoresis?

Presence of a specific antigen Signup and view all the answers

What is considered when defining the zones in immunodiffusion assays?

Antigen-antibody interaction levels Signup and view all the answers

What indicates that both reactants are present in a label free assay?

The formation of an Ab-Ag complex Signup and view all the answers

Which zone corresponds to the point where the concentration of Ag lies within the equality during immunodiffusion assays?

Equivalence zone Signup and view all the answers

In the Ouchterlony immunodiffusion assay, how long is the dish typically incubated?

24 - 72 hours Signup and view all the answers

What is a key factor that affects the size of precipitin rings in single radial immunodiffusion (sRID)?

Concentration of Ab in the agarose Signup and view all the answers

Which technique offers more rapid detection compared to single radial immunodiffusion?

Rocket immunodiffusion Signup and view all the answers

In the single radial immunodiffusion assay, how is the concentration of Ag in a sample related to the diameter of the precipitin ring?

Linear relationship Signup and view all the answers

What role do negatively charged Ag play in the Rocket immunodiffusion?

They migrate towards a positive charge Signup and view all the answers

What is the primary function of visualizing Ab-Ag complexes in immunoassays?

To detect the presence of both reactants Signup and view all the answers

Study Notes

Introduction to Immunoassays

Immunoassays are used to detect and measure antigens or antibodies.
Label-free assays detect antibody-antigen complexes by their ability to scatter light.
Visible complexes form due to Ab-Ag interaction.
A constant concentration of Ab can be used to measure specific Ag.
Alternatively, coated cells or particles bind to the target Ab or Ag, causing precipitation or agglutination.

Learning Objectives

Visualizing antibody-antigen (Ab-Ag) complexes
Understanding label-free assays
Defining pre-zone, equivalence-zone, and post-zone
Understanding the principles of immunodiffusion assays
Providing examples of immunodiffusion assays
Describing the Latex agglutination assay (LAA) procedure

Label-Free Assays - Equivalence

Pro zone: Antibody excess, little precipitation.
Zone of equivalence: Optimal amount of Ag and Ab, maximum precipitation.
Post zone: Antigen excess, little precipitation.

Immunodiffusion Assays

Ouchterlony Immunodiffusion Assay (Double Diffusion):
- Antibody (Ab) placed in center well, antigens in surrounding wells.
- Ab-Ag complexes form precipitin lines
- Patterns indicate similarities or differences between Ag.
- Patterns include identity, partial identity, and non-identity.
Single Radial Immunodiffusion (sRID) or Mancini Technique:
- The precipitin ring size depends on antigen concentration, antibody concentration, and sample volume.
- Rings form when Ag concentration is within the zone of equivalence.
- Precipitate diameter is proportional to antigen concentration (linear plot).

Activity - sRID Data

Example data tables for X-179A and X-187
Results for sRID assays including untreated, acid, etc

Immunodiffusion Assays - Electroimmunodiffusion

Rocket Immunodiffusion (Laurell Technique):
- Electrophorosis of the antigen(Ag) in an agarose gel containing antibody(Ab).
- The negatively charged antigen migrates towards a positive charge.
- Rocket height is proportional to the antigen concentration.

Immunodiffusion Assays - Immunoelectrophoresis

Investigation of proteins in a complex mixture to identify immunological characteristics.
Separating the protein samples by electrophoresis in circular wells.
Antiserum is placed in a central slot, antibodies precipitate proteins during diffusion.

Crossed Immunoelectrophoresis

First dimension: Separating proteins by electrophoresis.
Second dimension: Separated proteins further electrophoresed into a gel containing antibodies against the protein.
Precipitates form, peak or rocket shapes, area proportional to amount of Ag.

Latex Agglutination Assay (LAA)

Quick alternative to traditional immunodiffusion.
Samples (blood, saliva, cerebrospinal fluid (CSF))
Latex beads coated with antigen for antibody capture.
Steps include: preparation of standard and samples, serial dilutions, preparation of beads, sample loading, and spectrophotometer reading.
Total duration is two hours

The Hook Effect

High analyte concentrations inhibit complex formation, restricting sensitivity.
Leading to lower detection limits.

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Description

This quiz explores the basics of immunoassays, including the detection and measurement of antigens and antibodies. It covers label-free assays, the principles of immunodiffusion, and specific procedures like the Latex agglutination assay. Test your understanding of antibody-antigen interactions and the factors affecting precipitation.

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