saline. After introducing the reagents, the plates are left in a moisture chamber at room temperature. The results are preferably read after 2 days, although an approximate

 

Since the diffusion velocity of any molecule is dependent

on temperature, it is essential in using Oudin’s technique

that the temperature be kept strictly constant during the

reaction period.

The results of EID by Laurell’s method and single radial

immunodiffusion as described by Mancini, Carbonara and

Heremans are not significantly affected by temperature

fluctuations. One advantage of Oudin’s method is that the

solutions require less exact volume measurements, and

pipetting errors which may affect other methods are of

minor importance.

Single Radial Immunodiffusion

Principle: Dissolved antigen molecules diffuse radially

from a cylindrical well into an agar gel layer of uniform

thickness containing the corresponding antiserum. The

resulting precipitate assumes the form of a cylinder or

ring. When diffusion ceases, the surface area of the base

of the cylinder is directly proportional to the quantity of

antigen, at a predetermined concentration of antiserum in

the reaction gel.

If the circular area πr2

 (i.e. area of central well + area

of precipitate) is plotted on a graph as a function of the

quantity of antigen QAg (i.e. antigen concentration ×

volume of antigen solution), the result is a straight line

relationship expressed by the equation πr2

 = k × QAg + S.

The point S at which the straight line intercepts the

ordinate is a function of the size of the central well. And

the slope k is inversely proportional to the antiserum

concentration in the gel and the gel thickness. By

standardizing the technical conditions, it is possible to

keep constant the variables contained in k and S so that

the measured radius or diameter of the precipitate ring

is a function solely of the quantity of antigen introduced.

With the aid of standardized protein preparations, it is

Diagnostic Immunology 699

possible to construct a reference curve which can be used

to determine the antigen concentrations of unknown

solutions. It is essential, however, to deliver identical

volumes of the antigen solutions, standard and test

solutions, into central wells.

Differences in temperature do not affect the results of

this reaction. The only effect of a rise in temperature is

to accelerate diffusion and thereby the appearance of a

measurable precipitin ring. However, the temperature

should not be allowed to rise above 37oC because the gel

may melt and because of the risk of irreversible damage to

the thermolabile proteins.

Electroimmunodiffusion

Principle: In the course of the electrophoretic migration

of an antigen through agarose gel containing the

corresponding antiserum, it produces an extended trail of

an immunoprecipitate. The length ‘I’ of the precipitate is

a measure of the antigen concentration provided that the

latter is contained in a fixed volume of solvent.

Depending on the electrophoretic migration rate of

an antigen, the precipitin peaks may appear more or less

rapidly. Fast moving proteins usually produce long narrow

precipitin bands ending in a point, while proteins of lower

eletrophoretic mobility produce broader precipitates

with rounded ends. Because of their slow migration rates,

elctrophoresis takes longer; diffusion of the antigen at right

angles to the direction of electrophoresis may be responsible for the broadening of the peaks. The migration velocity

of the antigens is affected by the field strength and the pH

of the buffer solution employed. Careful standardization

of these factors is essential for reproducible results.

This makes the method somewhat more elaborate than

comparable methods based on diffusion alone. On the

other hand, the results are usually available in 2 or 3 hours,

while the methods of Oudin and Mancini require at least

20–30 hours.

Single Radial Immunodiffusion

Equipment

Antiserum-agar gel plates of single radial immunodiffusion

by the method of Mancini, Carbonara and Heremans as

modified by Augener can be prepared in polystyrene Petri

dishes (8 cm diameter) having perfectly flat bottoms.

Application of exact amounts of antigen samples is best

achieved by the use of a microliter syringe with which

exact amounts of 1 µL can be delivered.

The immunoprecipitin rings can be measured with a

measuring microscope or a magnifying lens with 0.1 mm

scale. A simple way of measuring is by using a measuring

template (with Tripartigen plates from Behring Diagnostics,

these are given as part of the kit for immunoglobulin, etc.

quantitations).

Procedure

A microliter syringe is used to fill holes in the agar gel

layer with 2 µL of antigen solution. At least three different

dilutions of each standard solution are required to plot

a curve. The remaining holes are used for the solutions

being analyzed. Each is filled with 2 µL of the test

specimen in suitable dilution; the diluent is physiological

saline. After introducing the reagents, the plates are left

in a moisture chamber at room temperature. The results

are preferably read after 2 days, although an approximate

reading can be obtained in one day. After 2 days, the

circular immunoprecipitates in the gel layer are distinct

and can easily be measured. For Tripartigen (from Behring

Diagnostics) a Tripartigen ruler (scale) is provided to read

the ring diameter and the quantitation is done by noting

the corresponding value given in appropriate units from

the reference chart provided. With every kit, a standard

serum/specific protein solution can also be had for

comparing results.