Zone in which the optimum precipitation occurs

Number of multivalent sites of antigen and antibody are approximately equal

*For a precipitation reaction to be detectable, the reaction must occur in this zone

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

Formulated by Marrack Each antibody must have at least two

binding sites and antigen must be multivalent

False negative reaction may occur due to the presence of high concentration of antibodies

If the reaction is suspected to be false negative: dilute out antibody and perform the test again may produce a positive result

Excess antigen may obscure the presence of small amount of antibody

To correct postzone phenomenon: test is repeated with an additional patient specimen taken about a week later

Modification of single-diffusion

Used to measure IgG, IgM, IgA, and complement components

Already replaced by more sensitive and automated methods

Standard Curve

Precipitin Rings A B C

a b c

Standards Samples

Diameter = log of concentration -log of diameter

One reagent: Either antibody or antigen is fixed

Other reagent moves in only one directionThe antibody is immobilized in a gelThin layer of Antigen Diffuses downward into the gel

Antgien Solution

Precipitation of Antigen/Antibody

Complexes

Antibody is distributed in the gel, and antigen is placed in wells cut in the gel.

Used to facilitatemigration of the antigen to the agar.

Antigen diffuses out of the well, and precipitation begins.

Precipitin line that is conical in shape,

resembling a rocket

Height of the rocket - Measure from the

well to the apex

Directly in proportion to the

amount of antigen in the sample.

Much more rapid than RID

Results can be obtained in a few

hours

It is essential, however, to

determine the net charge of the

molecules at the pH used for the test because this determines the

direction of migration within

the gel.

Using a buffer of pH 8.6

Other applications Too low to be detected

by nephelometry

Too high for RID

Agar or agarose gel mediumEquivalence ratioPrecipitation of immune complexParallel wellsPrecipitin lineNot widely use

•An immunochemical technique wherein both antigen and antibody diffuse independently through a semisolid medium in two dimensions, horizontally and vertically.

•Principle: Double Diffusion

Agar gel- usual transport medium

Incubation period- 12 to 48 hours

Zone of equivalence-Point of maximal precipitation

Ouchterlony plates

Different patterns possible:1.) Serologic identity

Fusion of the lines at their junction to form an arc represents serologic identity or the presence of a common epitope.

2.) NonidentityPattern of crossed lines demonstrates two separate reactions and indicates that the compared antigens share no common epitopes.

3.) Partial identityFusion of two lines with a spur.

Uses of this technique

- Identification of fungal antigens- Detection of antibodies to extractable nuclear antigens.

Irregular patterns maybe due to:

- Overfilling the wells- Irregular hole punching- Nonlevel incubation

Other factors affecting the accuracy of results:

- Drying out of the gel- Inadequate time for diffusion- Resulting in weakness of band intensity- Fungal or bacterial contamination of the gel

•Introduced by Grabar and Williams in 1953

•Double diffusion technique that utilizes an electric current to enhance results

• Performed as a two-step process and can be used for semiquantitation of a wide range of antigens

• Source of antigen is usually the serum, which is electrophoresed to separate out the main protein fraction

•Also used as a screening tool for the differentiation of more than 30 serum proteins including the major classes of immunoglobulins

Immunoelectrophoresis is a combination of electrophoresis and immunodiffusion

•Used in the clinical laboratory for the detection of myelomas, Waldenstrom’s macroglobulinemia, malignant lymphomas, and other lymphoproliferative disorders

• Also detects immunodeficiencies and deficiencies of complement components

• Replaced by immunofixation electrophoresis which gives quicker results and is easier to interpret

• Diagnosis of monoclonal gammopathy and in the urine is the demonstration of Bence Jones Proteins 

Fig. 1 Urine immunoelectrophoresis

If BJ protein is present in a urine specimen, precipitin lines will form with either κ (kappa) or λ (lambda) anti-L chain antisera because BJ protein is composed of homogenous L chains of a single antigen type, either κ or λ.

SOURCES OF ERROR IN ELECTROPHORESIS

Turbidity

Ability of particles in suspension to refract and deflect light rays passing through the suspension such that the light is reflected back into the eyes of the observer.

measured through optical density (OD)spectrophotometer or colorimeter

Turbidimetry

A method for determining the concentration of a substance in a solution by measuring the loss in intensity of a light beam through a solution that contains suspended particulate matter.

Antigen and antibody are mixed

The initial turbidity will form a precipitate which is then quantified.

Device used to measure the light passing through a solution

Placed at an angle of 180 degrees from incident light

If light absorbance is insignificant: turbidity can be expressed as absorbance which is related to the concentration of suspended particles and path length

Measures low-level antigen-antibody reactions

Detect if there is a presence of contaminationQuantification of serum proteins,

complement components Detect the presence of either an antigen or

antibody

All reagents or sera to be used must be free of particles that could scatter the light

Polyethylene glycol

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

Amount of light scattered is an index of the concentration of the solution

Nephelometers Measure light scatter at angles ranging from 10°

to about 90°Preferred method for measurement of low-level-

antigen-antibody reactionsEnd point nephelometry

Reaction is allowed to run essentially to completion

Large particles tend to fall out of solution and decrease the amount of scatter

Kinetic or Rate nephelometryRate of increase of scattering is measured

immediately after the reagent is addedRate change is directly related to antigen

concentration if the antibody concentration is kept constant

APPLICATIONS OF NEPHELOMETRY

Quantification of serum proteins

IgG, IgA, IgM and IgEComplement components C3, C4 and C1

inhibitorHaptoglobin, C-reactive protein,

transferrin, albumin, alpha1-antitrypsinApha2-macroglobulin, fibrinogen,

ceruloplasmin and rheumatoid factor

COMPARISON OFPRECIPITATION TECHNIQUES

IMMUNODIFFUSION TECHNIQUES