intersect forming so-called “spurs” (e.g. γ G-globulin) and γ A-globulin). Immunological non-identity is reflected by intersection of the precipitin lines without fusion (e.g. albumin and γ G-globulin).

To date, approximately 30 different precipitin lines in

human serum can be detected by immunoelectrophoresis

(IEP), indicating an equal number of individuals serum

proteins. Of late, many of these serum proteins have been

characterized.

The immunoelectrophoretic technique is a valuable

addition to available methods for characterization of

proteins. This technique is usefully employed in all

areas of protein chemistry and in connection with the

investigation of a large number of clinical problems

connected with protein metabolism. In particular,

the microtechnique of IEP has found widespread

acceptance.

694 Concise Book of Medical Laboratory Technology: Methods and Interpretations

Methodology

Double Diffusion of Ouchterlony

Macrotechnique

Reagents

1. Phosphate buffer solution 0.15 mol/L, pH 7.1. This

solution is obtained by mixing 67 mL of solution I with

33 mL of solution II.

Solution I: Distilled water is added to 26.70 g of Na2

HPO4. 2H2O to total 1,000 mL.

Solution II: Distilled water is added to 20.41 g of KH2PO4

to total 1,000 mL.

2. Agar

 Behringwerke agar purum or agar of equal quality

which possesses satisfactory transparency.

3. Antisera

 Antisera to plasma proteins.

Preparation of Agar Gel Slides

Four grams of purified agar is mixed with 100 mL of

phosphate buffer solution 0.15 mol/L, pH 7.1, and 300 mL

of distilled water. Approximately 40 mg of Thimerosal are

added as a preservative. This mixture is heated at 100oC

until the solution is clear. Undissolved particles can be

removed by centrifugation of the hot solution at 3,000 rpm

for 2–3 minutes.

Twenty two milliliters of the hot agar solution is then

pipetted into level Petri dishes of 8 cm diameter. Upon

standing for a short time, the agar solution will solidify,

forming a gel. Holes (wells) are cut into the agar gel by

means of a glass or metal punch. For routine examinations, it is best to cut holes of 0.6 to 0.7 cm in diameter

approximately 2 cm apart (as measured from the center

of the holes). Punch sets of various patterns are available

commercially. After cutting the holes, a small amount of

agar gel is heated with several drops of water in a test tube.

A few drops of this diluted agar solution are added to each

hole to seal the gaps between the agar gel and the bottom

of the Petri dish.

Gel Diffusion Procedure

Petri dishes prepared in this manner can be used for many

types of immunoprecipitation reactions. Multivalent antisera

(e.g. antiserum to human serum), heterogeneous antigen

solution (e.g. human serum), purified proteins (human IgG,

human IgA, or human albumin), or specific antisera (e.g.

antisera to plasma proteins) can be analyzed by this method.

The wells in the agar plate are filled completely with

approximately 0.1 mL of antigen solution or antiserum.

Highly precipitating antisera react optimally with antigen

solutions of concentrations between 1 and 10 mg/mL. The

Petri dish is covered to prevent drying of the agar, then

allowed to stand at room temperature for 2–3 days.

During this period, immunoprecipitates form. Each

individual antigen produces a single precipitin line, the

location of which depends upon the rate of diffusion and

the concentration of the antigen. The diffusion rate of

protein depends primarily upon the molecular weight.

High molecular globulins (e.g. macroglobulins) are

precipitated in the proximity of the point of application,

in contrast to antigens of lower molecular weight (e.g.

albumin) which are precipitated nearer to antibody well.

Immunologically, identical proteins in adjacent wells

form precipitin lines which merge completely (“reaction

of identity”). Partial immunological identity (partially

shared antigenic determinants) is reflected by formation

of precipitin arcs, which partially fuse and partially

intersect forming so-called “spurs” (e.g. γ G-globulin) and

γ A-globulin). Immunological non-identity is reflected

by intersection of the precipitin lines without fusion (e.g.

albumin and γ G-globulin).

Microtechnique

A microtechnique, using small agar gel plates, with smaller

Ag wells and shorter distances between them, is applicable,

under certain circumstances. Examples include the

need to examine small amounts of test material, or if test

results are needed in a short time period. Plates for this

microtechnique may be prepared with a 2% agar solution

as used for immunoelectrophoresis. Three mL quantities

of the hot agar solution are pipetted onto precleaned glass

slides (2” × 2”) and the agar is then allowed to solidify.

The agar wells are cut either individually or by means of

a template.