electrophoresed, since migration to the other side of the Ab trough is a possibility. Electrophoresis

 

Recording of Results

Photographic recording of results of gel diffusion test is the

most satisfactory method of documentation. Precipitin

lines can be copied directly onto photographic paper.

For this purpose, the surface of the agar gel is carefully

rinsed with tap water to remove dust or other undissolved

particles in the antisera or antigen solutions. To obtain an

optically uniform surface, the agar is covered by tap water

or physiological saline solution. The Petri dish is then

placed on photographic paper (extra hard) in a dark room

and illuminated from above with a lamp of approximately

200 watts. When optimally exposed and developed, the

photographic paper will exhibit even fine precipitin lines

with great sharpness.

Staining of the precipitin patterns of gel diffusion preparations can be achieved by the methods described under

“immunoelectrophoresis” if this should prove necessary.

Diagnostic Immunology 695

Possible Sources of Error

1. Specific antisera to plasma proteins are obtained

by immunizing rabbits or goats with highly purified

plasma proteins. Frequently, traces of contaminating

antibodies in such antisera must be removed by

absorption. Absorbed antisera may contain minute

amounts of antigens (human plasma proteins) used

for this purpose. This possibility must be considered

when precipitin lines occur between the antisera wells,

where two or more types of antisera are used.

2. Occasionally, circular precipitates develop around the

antigen wells. These “hallos” occur particularly when

markedly lipemic or aged samples of sera or plasma

are used. These nonspecific precipitations are easily

distinguished from specific immunoprecipitates by

their circular arrangements. Strongly hemolytic sera

may mimic nonspecific precipitates on photographic

records.

Immunoelectrophoresis

Reagents and Equipment

Agar

Agar used for IEP should have a low calcium concentration

and should yield a transparent gel with a low solidification

point. Agar of good quality can be prepared by washing

commercially available agar. It is important to wash agar

thoroughly with distilled water since electrophoretic

separation of serum proteins depends partly upon the

purity of agar. For example, in unwashed agar the protein

migration to the anode in the electrical field is limited;

the majority of proteins migrate to the cathode due to

the marked electro-osmotic potential. Agar specifically

prepared for IEP is available from Behring diagnostics.

Particularly suitable as a carrier medium for IEP is the

low-ion agarose, which is obtained by the removal of the

agaropectin moiety of agar.

Buffer

Michaelis diethylbarbiturate acetate buffer solution pH =

8.2, µ = 0.1.

A total of 13.38 g of sodium 5, 5-diethylbarbiturate

and 8.83 g of sodium acetate trihydrate are dissolved in

distilled water to yield 1.5 liters. The pH is adjusted to 8.2

by adding approximately 180 mL 0.1 N hydrochloric acid.

This solution possesses an ionic strength of µ = 0.1.

Antisera

Depending upon the specific problem to which the

technique is being applied (e.g. IEP analysis of human

serum) either the multivalent antisera (i.e. antisera

containing numerous antibodies, such as those directed

against the various proteins of human serum) or the

specific antisera (i.e. antisera directed against specific

antigens, such as albumin, IgG, etc.) can be employed.

Such antisera can be prepared in horses, rabbits, goats

chickens, or other suitable animals. The higher the

immunoprecipitation titer of the antiserum (as shown by

the ring test), the stronger are the precipitin lines on IEP.

With rabbit antiserum to human serum, approximately

30 different human serum proteins can be demonstrated

by IEP. A somewhat different immunoelectrophoretic

pattern is obtained with horse antihuman antiserum.

This difference is due to an altered Ag-Ab ratio which

is dependent upon the species specific differences of

antibody structures. Rabbit antisera form strong precipitin

lines in a wide range of Ag-Ab ratios, whereas horse

antisera produce finer and sharper precipitin lines in a

rather narrow range of Ag-Ab ratios. For this reason, it may

be advisable to use various dilutions when using horse

antisera.

Preparation of Agar Gel Slides

Two grams of pure agar is dissolved in 50 mL of

diethylbarbiturate acetate buffer, pH 8.2 (µ = 0.1) (can

check by pH meter), and 50 mL of distilled water. This

solution is heated for 15 minutes in a water bath of 100oC.

Any undissolved particles can be removed from the heated

agar by centrifugation at 3,000 rpm for 2–3 minutes. Ten

mg of Thimerosal is added as a preservative. The hot agar

solution is applied by pipette to alcohol cleansed, level

glass slides. 3 mL of agar are placed onto each glass slide.

After a few minutes the agar solidifies and the glass slides

thus prepared are put into moisture chamber. A simple

plastic container with Petri dish of water could serve as a

moisture chamber.

It is advisable to prepare just sufficient agar solution

to cover the glass slides since repeated heating of the gel

leads to alterations which interfere with electrophoretic

process. Holes and troughs are punched through the agar

layer with a die or suitable modification. On both sides of

the trough, a hole is made. Agar remaining in the punch is

aspirated by a syringe connected to the barrel of the punch

by rubber tubing.

Application of Antigens

The antigen mixture, e.g. serum, to be electrophoresed is

introduced into the wells by a 26-gauge needle attached

to a tuberculin syringe or by means of an appropriate

micropipette. Complete fillings of the wells require about

0.002 mL of serum or protein solution. Each slide provides

696 Concise Book of Medical Laboratory Technology: Methods and Interpretations two Ag wells, permitting antigen analysis in duplicates or

comparison of two different antigens. For the analysis of

enzymatically cleaved agents or substances which diffuse

readily, it is recommended that only one antigen be

electrophoresed, since migration to the other side of the Ab

trough is a possibility.

Electrophoresis

In principle, the apparatus used for IEP is similar to that

used for paper electrophoresis. The ends of the antigencontaining agar covered microscope slides are placed on

two parallel bars. Filter paper strips establish the contact

between the agar on the slides and the buffer in the

troughs. The slides must be horizontal in position. The

voltage between the ends of the slides is 45 V (6 volts/cm).