PRECIPITATION

Kraus

First noted the precipitation

Precipitation

Involves combining soluble antigen with soluble antibody (precipitin) to

produce visible insoluble complexes

Precipitation

o Rapid invisible formation of antigen-antibody aggregates

Precipitation

o Simplest method for detection of antigen-antibody reactions

IgG(IgG3)

Best antibody for precipitation

Affinity

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

antibody

Paratope is for?

Antigen

Epitope is for?

1. Ionic bond

2. hydrophobic bond

3. hydrophilic bond

4. Van der Waals force

Affinity:

Avidity

o Sum of all attractive forces between and antigen and an antibody.

Avidity

o It represents the overall strength of antigen–antibody binding

Law of Mass Action

All antigen–antibody binding is reversible and is governed by the ___

Law of Mass Action

This law states that free reactants are in equilibrium with bound reactants

Postzone

Antigen excess

Post zone

so insufficient reactive sites on antibody for lattice

formation • Every available antibody site is bound to a single antigen and

no cross-links are formed

Repeat the test after a week precipitation curve

Postzone Remedy

Prozone

Antibody excess

Prozone

so insufficient reactive sites on antigen for lattice

formation

perform Dilution precipitation curve

Prozone Remedy

Zone of Equivalence

Zone in which Optimum precipitation occurs

Zone of Equivalence

Each antibody or antigen binds to more than one antigen or antibody, respectively, forming a stable lattice

Precipitation In a fluid Medium

• Turbidimetry

• Nephelometry

Precipitation by passive immunodiffusion

• Oudin single diffusion

• Ouchterlony Double diffusion

• Radial immunodiffusion

Precipitation by electrophoresis

• Rocket immunoelectrophoresis

• Immunoelectrophoresis

• Immunofixation electrophpresis

Types of Precipitation Reaction

Turbidimetry

Measure of reduction in light intensity (light

blocked) using a spectrophotometer or

an automated clinical chemistry analyzer

Turbidietry

The amount of scatter is proportional to the

size, shape, and concentration of

molecules present in solution

Nephelometry

Measure of light scattering which indicates

the concentration of the solution

10 degrees to about 90 degrees.

Nephelometers typically measure light

scatter at angles ranging from _____

IgG,A,M,E

Antibody that detects by nephelmetry

Oudin Procedure

Ab is incorporated in agar gel in a test tube

Agar, Agarose, & Gel

Oudin Procedure : Support medium

Oakley-Fulthorpe

Antibody is incorporated in agar gel in a

test tube above which is placed a column

of plain agar and antigen is layered on

surface of this.

Oakley-Fulthrope

Antigen and antibody diffuse (double

diffusion) towards each other (in one

dimension) through the intervening column

of plain agar and forms a band of

precipitate where they meet at optimum

proportion

Radial Immunodiffusion

measures immunoglobulin concentration

Radial Immunodiffusion

As the antigen diffuses into the agar, it

combines with specific antibody to form

rings of precipitation (precipitin ring)

around the well

IgG- 24 hrs

IgM- 50-72 hrs or 2-3 days

Reading time:

• IgG-

• IgM-

Fahey and McKelvey/ Kinetic Mtd

Mancini/ End point Mtd

Radial Immunodiffusion:

Fahey and McKelvey / Kinetic Method

Uses measurements taken before the point

of equivalence is reached (about 19 hours)

Fahey and McKelvey / Kinetic Method

A graph is plotted using semi-log paper

with diameter on the x axis and

concentration on the y axis

Mancini / End point Method

Antigen is allowed to diffuse to completion,

and when equivalence is reached (occurs

between 24 and 72 hours), there is no

further change in the ring diameter

Mancini / End point Method

A graph is obtained by plotting

concentrations of standards on the x-axis

versus the diameter squared on the y-axis,

and a smooth curve is fit to the points

Ouchterlony Double Diffusion

Simplest and least sensitive method

Ouchterlony Double Diffusion

Semiquantitative analysis of concentration

of antigen or antibody

Ouchterlony Double Diffusion

Both antigen and antibody diffuse

independently through a semisolid medium

in two dimensions (horizontally and

vertically)

Ouchterlony Double Diffusion

Both antigen and antibody diffuse

independently through a semisolid medium

in two dimensions (horizontally and

vertically)

• Serological identity

• Non-identity

• Partial identity

Types of Ouchterlony

Serological identity

Fusion of the lines at their junction to form

an arc represents ____ or the

presence of a common epitope,

smooth curve

Serological identity forms a ___

Non-identity

Pattern of crossed lines demonstrates two

separate reactions and indicates that the

compared antigens share no common

epitopes,

cross-line

Non identity forms _____ pattern

partial identity

Fusion of two lines with a spur indicates_____

Partial identity

In this last case, the two

antigens share a common epitope, but

some antibody molecules are not captured

by antigen and travel through the initial

precipitin line to combine with additional

epitopes found in the more complex

antigen

Rocket Immunoelectrophoresis

One-dimension electro-

immunodiffusion, an adaptation of

radial immunodiffusion

Laurell

Rocket Immunoelectrophoresis was developed by ?

Rocket Immunoelectrophoresis

It is a combination of RID +

electrophoresis

Immunoelectrophoresis

Grabar and Williams

A double-diffusion technique that

incorporates electrophoresis current to

enhance results. Introduced by ____

and ___

Immunoelectrophoresis

Identification of proteins in body fluids

Immunoelectrophoresis

Differentiation of polyclonal from

monoclonal elevations in gamma

globulin

Alper and Johnson

Immunofixation Electrophoresis was described by ?

Immunofixation electrophoresis

Similar to immuno-electrophoresis

except that after electrophoresis takes

place, antiserum is applied directly to

the gel’s surface rather than placed in

a trough.

Immunofixation Electrophoresis

This method is especially useful in

demonstrating those antigens present

in serum, urine, or spinal fluid in low

concentrations

Western blot.

One of the best-known adaptations of

this technique(Immunofixation electrophoresis) is the ___

• Applying current in the wrong direction

• Incorrect pH of the buffer

• Incorrect electrophoresis time

• Inappropriate concentration of antigen and antibody

• Amount of current applied

Sources of Error in Electrophoresis

Nephelometry

Light that is scattered at an angle is measured, indicating the amount of antigen or antibody present

Radial Immunodiffusion

Antigen diffuses out into gel that is infused with antibody. Measurement of the radius indicates concentration of antigen

Ouchterlony double diffusion

both antigen and antibody diffuse out from wells in a gel. Lines of precipitate formed indicate the relationship of antigens

immunoelectrophoresis

Electrophoresis of serum followed by diffusion of antibody from wells

Immunofixation electrophoresis

electrophoresis of serum followed by direct application of antibody to the gel