Fixation of the color: Wash briefly with distilled water, stain for 10 minutes in a 0.1% solution of Ni(NH4)2 (SO4)2, thereafter for 12 hours in 0.2% Ni(NH4)2(SO4)2. The specific color is bluish black.

 

Adequate electrophoretic separation is achieved within 45

minutes. Under these conditions the agar slides warm to

approximately 25–28oC. Therefore, the application of a lid

is necessary to prevent desiccation.

Application of Antiserum

Subsequent to electrophoretic separation, the agar strip

from the precut center trough is removed with a 19-gauge

injection needle attached to tuberculin syringe. The

amount of antiserum applied is approximately 0.04 to

0.06 mL. It is advantageous to vary the antigen and

antiserum concentrations. A shorter antibody trough can

be used to examine only one serum fraction (e.g. IgG)

in order to economize on antisera. After application of

antiserum, the slides are placed in a moisture chamber for

diffusion. The electophoretically separated proteins (i.e.

antigens) and the antiserum diffuse toward one another

and the homologous agents undergo an Ag-Ab reaction

by forming precipitin lines at the points of confluence.

Depending upon the concentration of antigens and

antisera, optimal immunoprecipitates are formed

between 20 hours and longer.

Possible Sources of Errors

1. Slides must be cleaned with chromic sulfuric acid.

2. The electrophoretic separation of the serum protein

must be within an optimal range. Too short or too

long a separation distance may be caused by faulty

buffer composition (pH, µ), by incomplete contact

between agar slides and filter paper strips, or by

improper voltage. It is important to maintain uniform

distribution of voltage (not of current) through the agar.

3. Wells of proper diameters and the correct distance

between the Ag well and Ab trough are decisive for

satisfactory IEP. Deviation from these specifications

may result in marked dislocations and/or distortion

of precipitin patterns.

4. The agar gel slides should be used within 2 or 3 days

after preparation and must be stored in moisture

containing sealed containers, preferably at 4oC.

5. The sera to be examined should be fairly fresh or

stored by freezing at –20oC. Bacterial contamination

or autocatalytic processes may result in marked

alterations of serum samples, leading to changes in

electrophoretic mobility, solubility, etc.

6. Normal control sera should be examined simultaneously with abnormal sera.

Fixation of Precipitin Patterns

Immunoelectrophoretic patterns can be recorded by

photographing the unstained or stained precipitin lines. For

most purposes, direct photography of the slide is sufficient.

Staining of the precipitin patterns is occasionally of

value for chemical characterization of the precipitin lines.

The agar slides are washed with physiological saline for

one or two days, in order to remove the nonprecipitated

protein from the agar. They are then covered by the filter

paper and dried completely either in the incubator at 37oC

(or in air at room temperature). After removal of the filter

paper, the agar slides are placed in 2% acetic acid solution

for approximately 5 minutes. The various staining techniques performed are as follows.

Protein Stains

Amidoschwarz (Amido black): 0.5% amidoschwarz 10B in

methanol-glacial acetic acid (9:1). Stain for 5–10 minutes,

then wash with methanol-glacial acetic acid (9:1), for

approximately 15 minutes.

Azocarmine: 0.5% azocarmine B in methanol-glacial acetic

acid (9:1). Stain for 15 minutes; wash with methanolglacial acetic acid solution (9:1).

Light green: 0.5% green SF in 5% trichloroacetic acid. Stain

for 1 hour; wash with 5% trichloroacetic acid solution.

Bromophenol blue: 0.1% bromophenol blue in HgC12-

saturated methanol. Wash with methanol. Green stain

results from acid solution, blue stain from alkaline solution.

Lipoprotein Stains

Oil red: 0.5% oil red O in 50% ethanol (filtered). Stain for

approximately two hours. Wash with 50% ethanol.

Sudanblack: Sudanblack (0.1%) is dissolved in 60% ethanol

at 37oC, with occasional stirring, during a 24 hours time

period. This solution is then filtered at 25oC and stored

in dark containers. Before use, 0.1 mL of a 30% sodium

hydroxide solution is added to 160 mL of the sudanblack

solution. After staining for 2 hours, the slides are then

washed using 50% ethanol.

Diagnostic Immunology 697

Peroxidase Reaction (for haptoglobin and hemopexin)

Benzidine: Dissolve 0.2 g benzidine in 100 mL of distilled

water, then add 0.5 mL glacial acetic acid and 0.2 mL

hydrogen peroxide 30%. When stained for 10–20 minutes,

the specific precipitates become dark greenish-blue in

color.

Fixation of the color: Wash briefly with distilled water,

stain for 10 minutes in a 0.1% solution of Ni(NH4)2 (SO4)2,

thereafter for 12 hours in 0.2% Ni(NH4)2(SO4)2. The specific

color is bluish black.

Ceruloplasmin Stain

p-Phenylenediamine: Solution must be prepared

immediately before use. Dissolve 21.6 mg of

p-phenylenediamine in 100 mL of sodium acetate buffer

solution (pH 5.7, µ = 0.1); add 10 mL of a solution consisting

of 0.65 g sodium azide in 1,000 mL of distilled water, and

warm to 37oC. Stain for 2 hours at 37oC. Wash twice for 2

hours in sodium acetate buffer (pH 5.7). This technique is

suitable for fresh samples only.