IMSE LEC (Finals) - Immunoassay Part 2 (Precipitation Reaction)

Immunoassay

This have been developed to detect either antigen or
antibody, and they vary from easily performed manual tests to highly complex automated assays.

Precipitation:

involves combining soluble antigen with soluble antibody to
produce insoluble complexes that are visible.

Agglutination

is the process by which particulate antigens aggregate to form larger complexes when a specific antibody is present.

IgM

Antibody involved in agglutination reaction

Agglutination Reaction

Is a particulate antigens in which they form a large complexes

Large complexes

Clumping of the cells under Agglutination Reaction, in line with RBC

Affinity and Avidity

Two types of Antigen-Antibody binding

Affinity

It is the initial force of attraction that exists between a
single Fab site on an antibody molecule and a single epitope or determinant site on the corresponding antigen.

Affinity

Types of noncovalent bond that holds antigen-antibody together.

Affinity

The strength of attraction depends on the specificity of antibody for a particular antigen.

Affinity

Antibodies are capable of reacting with antigens that are
structurally similar to the original antigen that induced antibody production.

Cross-reactivity

Antibodies are capable of reacting with antigens that are
structurally similar to the original antigen that induced antibody production. This process is known as what?

Avidity

It represents the sum of all the attractive forces
between an antigen and an antibody.

Avidity

This involves the strength with which a multivalent
antibody binds a multivalent antigen, and it is a
measure of the overall stability of an antigen-antibody
complex.

Avidity

A high ______ can actually compensate for a low
affinity.

Avidity

Stability of the antigen-antibody complex is essential
to detecting the presence of an unknown, whether it is
antigen or antibody.

relative proportions

In addition to the affinity and avidity of the
antibody involved, precipitation depends on
the ________ of antigen and antibody
present.

- Prozone
- Zone of equivalence
- Postzone

Three Phenomenon of Precipitation/Precipitin Curve

Zone of equivalence

In this phenomenon, the optimum precipitation occurs, in which the number of the multivalence sides of the antigen and antibody are approximately equal.

Prozone

In this phenomenon, the mechanism is the antibody excess in which, the antigen combines with only one or two antibody molecules and no cross linkages are formed.

Postzone

In this phenomenon, the mechanism is the antigen excess in which, it is not correlated with a lattice network form.

Postzone

In this phenomenon, every available antibody site is bound in single antigen and no crosslinks are formed.

Precipitin curve

This shows how the amount of precipitation varies with varying antigen concentration when the antibody concentration is kept constant

Prozone

Excess antibody is called the

Postzone

Excess antigen concentration is called

• Measurement of precipitation by light scattering
• Determination of Precipitation via Passive immunodiffusion techniques
• Determination of Precipitation via Immunoelectrophoretic technique

Methods to determine the precipitation reactions

- Turbidimetry
- Nephelometry

2 methods to be considered in the measurement of precipitation by light scattering

• Radial immunodiffusion
• Ouchterlony double diffusion or Ouchterlony double immunodiffusion

2 methods to be considered in the determination of precipitation via passive immunodiffusion techniques

• Rocket immunoelectrophoresis
• Immunoelectrophoresis
• Immunofixation electrophoresis
• Countercurrent immunoelectrophoresis

4 methods to be considered in the determination of precipitation via immunoelectrophoretic technique

Turbidimetry

This measures the unscattered light

Turbidimetry

is a measure of the turbidity or cloudiness of a solution.

Turbidimetry

A detection device is placed in direct line with the incident light.

Turbidimetry

Collecting light after it has passed through the solution.

Turbidimetry

It thus measures the reduction in light intensity due to reflection, absorption, or scatter.

reflection, absorption, or scatter

Turbidimetry measures the reduction in light intensity due to _______, ________, or ______.

Turbidimetry

It is recorded in absorbance units, a measure of the ratio of incident light to that of
transmitted light

Precipitation reaction

This deals with the precipitation of the fluid

Turbidimetry and nephelometry

Fluid can be measured using

Light scatter

This occurs in the proportion to the size, shape, and the concentration of the molecules present in the solution

Spectrophotometer or automated clinical chemistry analyzer

Measurement of light scatter can be made using this two

Turbidimetry

Has the intensity of light transmitter through the medium

180 degree angle

Unscattered light is measured at ________ angle from the incident-like theme

Nephelometry

This measure the scattered light

Nephelometry

measures the light that is scattered at a particular
angle from the incident beam as it passes through a suspension.

• End-point nephelometry
• Kinetic or rate nephelometry

Two types of Nephelometry

Nephelometry

Many automated instruments utilize this principle for the measurement of serum proteins.

Nephelometry

provides accurate and precise quantitation of
serum proteins, and due to automation, the cost per test is
typically lower than other methods.

Nephelometry

Index of the solution concentration

Nephelometry

Can detect antigen and antibody

Antibody and patient sample

Reagents to detect the unknown antigen

End-point nephelometry

Reaction is allowed to run essentially to completion, but large particles tend to fall out of solution and decrease the amount of scatter.

Kinetic or Rate Nephelometry

rate of scattering increase is measured immediately after the reagent is added

Gel

The precipitation of antigen-antibody complexes can also
be determined in a support medium such as a

seaweed, agarose, and a purified agar

Gel is a high-molecular-weight complex polysaccharide
derived from ______, ______, and ______.

rate of diffusion

This is affected by the size of the
particles, the temperature, the gel viscosity, and the
amount of hydration.

Agarose and purified agar

What are the most used material which are consist in a gel because of the stabilization and visualization

Visualization of the Precipitate bonds

Positive reaction in diffusion process

James Oudin

He was the first to use gels for precipitation reactions, and he pioneered the technique known as single diffusion.

Radial Immunodiffusion (RID)

antibody was incorporated into agarose in a test tube

Radial Immunodiffusion (RID)

The antigen was layered on top, and as the antigen moved down into the gel, precipitation occurred and moved down the tube in proportion to the amount of antigen present.

precipitation

The antigen was layered on top, and as the antigen moved down into the gel, ________ occurred and moved down the tube in proportion to the amount of antigen present.

Radial Immunodiffusion (RID)

A modification of the single-diffusion technique was the _______

Radial Immunodiffusion (RID)

antibody is uniformly distributed in the support gel, and antigen is applied to a well
cut into the gel.

Radial Immunodiffusion (RID)

As the antigen diffuses out from the well, antigen- antibody combination occurs in changing proportions until the zone of equivalence is reached and a stable lattice
network is formed in the gel.

stable lattice network

In Radial Immunodiffusion (RID), as the antigen diffuses out from the well, antigen- antibody combination occurs in
changing proportions until the zone of equivalence is reached and a ________ is formed in the gel.

Radial Immunodiffusion (RID)

The area of the ring obtained is a measure of antigen concentration, and this can be compared to a standard curve obtained by using antigens of known concentration.

A) Mancini method
B) Fahey and Mckelvey method

Two techniques for the measurement of Radial Immunodiffusion (RID)

antigen concentration

In Mancini method, the DIAMETER OF THE RING is equivalent in ________

End point method

What method use in Mancini method

Mancini method

In this method/technique for the measurement of Radial Immunodiffusion (RID), antigen is allowed to diffuse to completion

IgM and IgG

Mancini method is used for the determination of this two immunoglobulin

50-72hrs

Mancini method:
• Determination of IgM: reaction is completed at _______

24 hours

Mancini method:
• Determination of IgG: reaction is completed at _____

Fahey and Mckelvey method

In this method/technique for the measurement of Radial Immunodiffusion (RID), the diameter is proportional to the log of the concentration.

18 hours

Reactions of Fahey and Mckelvey method is measured after

Kinetic method

What method use in Fahey and Mckelvey method

- Overfilling/underfilling the wells
- nicking the side of the wells when filling
- spilling sample outside the wells
- improper incubation time and temperature, and
- incorrect measurement

Sources of error of Radial Immunodiffusion (RID)

Radial immunodiffusion

This has been used to measure IgG, IgM, IgA,
and complement components.

IgG, IgM, IgA, and complement components

Radial immunodiffusion has been used to measure _____

Radial immunodiffusion

It is simple to perform and requires no instrumentation, but it is fairly expensive to run.

Radial immunodiffusion

This has largely been replaced by more
sensitive and automated methods such as nephelometry and enzyme-linked immunosorbent assays except for low volume analytes such as IgD or IgG subclasses.

Ouchterlony Double Diffusion

In this technique, both antigen and antibody diffuse independently through a semisolid medium in two dimensions, horizontally and vertically.

12 and 48 hours in moist chamber

Incubation of Ouchterlony Double Diffusion

Precipitin lines

What will form in Ouchterlony Double Diffusion

precipitin lines

In Ouchterlony Double Diffusion, _________ form where the moving front of antigen meets that of antibody

Central well

In Ouchterlony Double Diffusion, antibody that is a mixture of anti-1 and anti-2 is placed in the ________

Outside walls

In Ouchterlony Double Diffusion, the unknown antigens are placed in the ______

- Serological identity
- Nonidentity
- Partial identity

In Ouchterlony Double Diffusion, what are the three identities?

Serological identity

This identity, the arc indicates that the two antigens are identical

Nonidentity

This identity, two crossed lines represent two different precipitation reactions. The antigens share no identical determinants.

partial identity

This identity, antigen 1a shares a determinant that is part of antigen 1, but it is not as complex.

Spur

In Ouchterlony Double Diffusion, the _____ formed always points to the simpler antigen.

rocket immunoelectrophoresis

This is also known as laurel technique

Laurel technique

Rocket Immunoelectrophoresis is also known as

antibody, antigen

In Rocket Immunoelectrophoresis, this _______ (reagent) is distributed in the gel and _____ (sample) is added.

conical shape precipitin line

What is the end result in Rocket Immunoelectrophoresis

1 to 3

In Rocket Immunoelectrophoresis, standards are in wells ________

4 to 6

In Rocket Immunoelectrophoresis, patient samples are in wells

4

In Rocket Immunoelectrophoresis, well ____ contains no antigen, because no ring is formed.

5, 6

In Rocket Immunoelectrophoresis, well ____ has a low concentration of antigen, and well _____ has a high concentration of antigen.