Antigen-Antibody Interaction, Precipitation, Agglutination Part 2

What is precipitation?

Soluble antigen combines with soluble antibody to form an insoluble precipitate.

What are light scatter techniques used to detect?

Large antigen-antibody precipitate complexes in solution.

What are the two things that can happen to light when it passes through a solution?

Light can either be transmitted through the solution or scattered by the complexes.

What is turbidimetry?

A technique that measures light transmitted directly through a solution to measure cloudiness.

What causes cloudiness in turbidimetry?

The size

How does turbidimetry detect complexes?

Light passes through the solution and transmitted light is measured.

How is turbidimetry reported?

In absorbance units.

What is nephelometry?

A technique that measures light scattered at an angle by particles in a solution.

Where is the detector in turbidimetry?

In the direct path of the light.

Where is the detector in nephelometry?

At an angle to the direct light path

What is the relationship between scattered light and concentration?

The amount of scattered light is proportional to concentration.

How is nephelometry reported?

In arbitrary units.

Which is more sensitive: turbidimetry or nephelometry?

Nephelometry.

What do turbidimetry and nephelometry both provide?

Quantitative values using a standard curve.

What is a standard curve?

A graph made using samples with known concentrations to relate signal to concentration.

What are standards?

Samples with known concentrations used to create a standard curve.

How are standards different from controls?

• Standards calculate patient concentrations

• Controls check that the test is working properly.

How is a standard curve used?

The patient's signal is compared to the curve to determine concentration.

What is passive diffusion?

Antigen and antibody diffuse through a medium

Why is gel used in passive diffusion?

It stabilizes the movement of antigen and antibody.

What happens when antigen and antibody diffuse through the gel?

They encounter each other and can reach the zone of equivalence.

What happens at the zone of equivalence during diffusion?

An insoluble precipitate forms.

What can the precipitate look like in diffusion?

A line or ring.

What is single diffusion?

One substance is already in the gel and the other is added to a well.

How many wells are used in single diffusion?

One well.

What is already in the gel during single diffusion?

Either antigen or antibody.

What is double diffusion?

Both antigen and antibody are added to separate wells and diffuse through the gel.

How many wells are used in double diffusion?

Two wells.

What is known in double diffusion?

One substance is known and the other is unknown.

What is Radial immunodiffusion (RID)

A single diffusion technique used to quantitatively measure antigen concentration.

What is already in the gel in RID?

Known antibody.

What type of antibody is used in RID?

High-affinity, monoclonal

What is added to the well in RID?

Patient sample containing antigen.

What happens to antigen in RID?

It diffuses outward through the gel.

What forms at the zone of equivalence in RID?

A precipitate ring.

What does the ring size in RID indicate?

The concentration of antigen.

Is RID qualitative or quantitative?

Quantitative.

How is concentration determined in RID?

Compare the patient's ring size with known standards.

What are the two types of RID?

• End-point RID

• Kinetic RID.

What is End-point RID (Mancini)?

Antigen diffuses until the reaction reaches completion.

How long does end-point RID take?

About 24–72 hours.

What is proportional to antigen concentration in end-point RID?

The square of the ring diameter.

What is Kinetic RID (Fahey and McKelvey)?

Measurement is taken before the point of equivalence is reached.

Why is kinetic RID faster?

It does not require antigen to diffuse to completion.

How long can kinetic RID take?

About 18 hours.

What is proportional to antigen concentration in kinetic RID?

Ring diameter is proportional to the log of antigen concentration.

What are the clinical applications of RID?

Quantitative assays for immunoglobulins and complement.

What are advantages of RID?

Simple to perform.

What are disadvantages of RID?

Expensive gels and time-consuming.

What is Ouchterlony double diffusion

A double diffusion technique where antigen and antibody form precipitin lines in a gel.

What is placed in the center well in Ouchterlony?

Polyclonal antibody.

Why is polyclonal antibody used in Ouchterlony?

It can bind multiple epitopes.

What is placed in the outer wells of Ouchterlony double diffusion?

Known antigens and the patient antigen.

Why are known antigens used in Ouchterlony?

They are controls used to compare with the patient antigen.

What do you look for in Ouchterlony?

Precipitin lines and whether the patient's pattern matches the known antigen.

What does serological identity mean?

Patient antigen and known antigen are the same.

What happens to lines with serological identity?

The precipitin lines fuse together.

What does non-identity of serological mean?

Patient antigen and known antigen are not the same.

What happens to lines with non-identity?

The precipitin lines cross but do not fuse.

What does partial identity mean in serological?

Antigens share some epitopes but are not identical.

What happens to lines with partial identity?

Lines partially fuse and form a spur.

What does the spur point toward?

The simpler antigen.

What are active diffusion techniques?

Techniques that use electrical current through a gel to separate molecules.

What does electrical current do in active diffusion?

Separates molecules based on differences in electrical charge.

Can active diffusion be used with single and double diffusion?

Yes.

What are advantages of active diffusion

• Speeds up the reaction

• Increases clarity of precipitation.

What is a Rocket immunoelectrophoresis?

A technique similar to RID that uses electrical current to move antigen through antibody-containing gel.

What is in the gel during rocket immunoelectrophoresis?

Antibody.

What is placed in the well during rocket immunoelectrophoresis?

Patient antigen.

What does electrophoresis do in rocket immunoelectrophoresis?

Facilitates antigen migration through the gel.

What shape does the precipitin line form?

A rocket shape.

Is rocket immunoelectrophoresis quantitative?

Yes.

What is proportional to antigen concentration in rocket immunoelectrophoresis?

The height of the rocket.

How is concentration determined in rocket immunoelectrophoresis?

Use known standards to create a standard curve and compare the patient sample.

What is Immunoelectrophoresis (IEP)?

Patient and control samples that are separated by electrophoresis

What happens first in IEP?

Patient and control samples are separated by electrophoresis.

What is done after electrophoresis in IEP?

A trough is cut between the samples and antiserum is added.

Does antiserum undergo electrophoresis in IEP?

No

What forms after antiserum diffuses in IEP?

Precipitin lines or arcs.

What is IEP used for?

Comparing the patient's precipitation pattern with a control.

Is IEP qualitative or quantitative?

Qualitative and semiquantitative.

What do you look for in IEP?

Whether precipitin lines are similar

Is IEP commonly used clinically?

No

What is a disadvantage of IEP?

It is difficult to interpret.

What is Immunofixation electrophoresis (IFE)?

Separates patient proteins by electrophoresis and identifies specific proteins using antibodies.

What is added to the wells in IFE?

Patient antigen/protein sample.

What happens to patient proteins in IFE?

They are separated by electrophoresis.

What happens after proteins are separated in IFE?

The sample is fixed in place.

What is added to the individual wells in IFE?

Specific antisera

What happens when antiserum binds its target in IFE?

An insoluble immune complex forms.

What happens after immune complexes form in IFE?

The gel is washed and stained for visualization.

What antibodies can be used in IFE?

Anti-human serum

What is IFE used to investigate?

Changes or abnormalities in a patient's antibodies.

What is in the first well in the IFE example?

All proteins in the patient's serum.

What is in the IgG well?

Only IgG is detected.

What is in the IgA well?

Only IgA is detected.

What is in the IgM well?

Only IgM is detected.

What does mainly IgG in an IFE pattern indicate?

The abnormal antibody is primarily IgG.

What does monoclonal IgG gammopathy with lambda light chains mean?

An abnormal monoclonal IgG antibody is present with lambda light chains.