Immunoelectrophoretic analysis

3.7 Immunoelectrophoretic analysis

Studying antibody–antigen interaction solely by simple diffusion is possible if there are only a few components in the system but, if there are multiple antigens reacting with several antibodies, the precipitin lines become difficult to resolve and impossible to interpret.

Increased resolution can be obtained by combining electrophoresis with immunodiffusion in gels, in the technique known as immunoelectrophoresis. This is useful in the immuno- logical examination of serum proteins: Serum proteins separate in agar gels, under the influence of an electric field, into albumin and α1-, α2-, β- and γ-globulins. If you are not familiar with this electrophoretic separation of serum proteins, it is advisable to perform a simple agar gel electrophoresis as this will aid your understanding of the patterns obtained with the later techniques.

3.7.1 Agar gel electrophoresis

MATERIALS AND EQUIPMENT 2% agar in barbitone buffer

Barbitone buffer Precoated microscope slides Normal and myeloma sera 10% v/v glacial acetic acid in water Electrophoresis tank and power pack, e.g. BioRad; Shandon Scientific Ltd Gel punch

METHOD

1 Melt the agar in a microwave oven.

2 Mark the end of the slide that will be positive during the electrophoresis. If required, number the slides.

3 Pour 3–5 ml of agar onto the slide on a levelled surface.

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3.7IMMUNOELECTROPHORETIC ANALYSIS

4 When the agar has set, punch the pattern. (Smaller wells than used for immunodiffusion are required. A fine Pasteur pipette or a hypodermic needle with a square cut end may be used.)

5 Suck out the agar plugs.

6 Fill the wells with serum to which a small amount of bromophenol blue dye has been added.

7 Fill the electrophoresis tank with full-strength barbitone buffer.

8 Place the slide in the electrophoresis tank and connect each end of the slide to the buffer chambers with rayon or filter paper wicks. Close the tank.

9 Apply a current of about 8 mA/slide. The voltage drop will be about 5 –7 V/cm. Note: The bromophenol blue dye binds to the serum albumin and as this is the fastest migrating band it serves as a marker throughout the electrophoresis. If excess dye has been added, however, a bright blue band of free dye will run in front of the albumin towards the anode.

10 When the albumin band (blue) nears the end of the slideaafter about 60 minaremove the

slide and fix the proteins by immersing the slide in 10% glacial acetic acid.

11 Cover the slide with fine filter paper and leave to dry.

12 Dampen the paper and remove, then stain the slide with Coomassie brilliant blue.

Suggested design One well should contain normal serum and the other serum from a patient with multiple myelo-

matosis (a disease in which a single clone of antibody-forming cells becomes malignant and produces large amounts of monoclonal antibody). If you are using mouse reagents, then ascitic fluid or serum from a hybridoma-bearing animal will do equally well.

Results The main serum proteins should show clearly as oval bands. Identify each band (albumin, α1-,

α 2-, β- and γ-globulins), and assign the abnormal monoclonal band to one of these (see Fig. 3.9). TECHNICAL NOTE

The negative charge on the agar generates an electroendosmotic flow of water through the gel. This flow, and not the potential difference, is responsible for most of the separation seen with some of the globulins, which are near their isoelectric point under the conditions used. This is discussed in more detail later (see Section 3.7.2).

3.7.2 Immunoelectrophoresis

This is a powerful analytical technique with great resolving power, combining prior separation of antigens by electrophoresis with immunodiffusion against an antiserum (see Fig. 3.10).

MATERIALS AND EQUIPMENT As for agar gel electrophoresis but in addition: Anti-human whole serum

96 C H A P T E R 3: Antibody interactions with antigens

Fig. 3.9 Electrophoresis of serum

(a)

samples. (a) Agar gel electrophoresis. Well 1, normal serum; well 2, serum from patient with multiple myeloma. Myeloma patients show an overproduction of antibody, usually of

a single clone, in this case running in

the γ-globulin region. You can see by 1 inspection that there is an apparent

(b)

decrease in the albumin content of the myeloma serum. (b) Electrophoresis on cellulose acetate membranes. Sample 1,

normal serum as in (a); sample 2, myeloma serum as in (a); sample 3,

serum from another patient with multiple myeloma. The principle of this separation of serum proteins is basically

similar to that described for agar gel electrophoresis, except that the sample

Albumin α 1 α 2 β γ

is applied onto the membrane as a band rather than via a well. The advantages of this technique are: (i) it is easier to

(c)

discern the individual protein bands;

γ and (ii) it is possible to clear the

Albumin

membrane with either glycerol or one of the commercially available clearing

oils. Thus, the protein content of each band can be determined by scanning

photometry. The ‘hawk-shaped’ band seen in sample 3 is often observed with

Absorbance

myeloma protein and is probably

caused by overloading of this band.

(c) The traces obtained from scanning samples 1 and 2 of (b) are shown here.

By integrating the area under each peak (this is usually done automatically by

the scanner) it is possible to determine the total protein content of each band,

and so confirm the observation made in (a), that there is indeed an albumin :

Absorbance 0.4

γ -globulin reversal in the myeloma serum. Sample 1: normal serum, above

albumin : globulin ratio 4.5. Sample 2:

myeloma serum, below albumin :

globulin ratio 0.7.

METHOD

1 Prepare slide as for agar gel electrophoresis.

2 Cut the pattern shown in Fig. 3.11. (Although cutters and moulds are available commercially for many different patterns, the holes can be made with hypodermic needles (cut square and sharpened) and the trough with razor blades.)

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3.7IMMUNOELECTROPHORETIC ANALYSIS

3 Suck out the agar wells but do not remove the agar from the trough as this may cause abnormalities in protein banding during electrophoresis.

4 Fill one well with normal human serum and the other with myeloma serum.

5 Electrophorese as before.

6 Remove the agar trough and fill with anti-whole human serum.

7 Leave the slide to incubate overnight in a humid chamber at a constant temperature. (Again, lines will appear within 2–3 h if the slide is incubated at 37°C.)

8 Examine the lines produced and identify the IgG, IgA and IgM bands, and the bump in the precipitation arc typical of monoclonal immunoglobulin in the myeloma serum. The result obtained should be similar to that shown in Fig. 3.11.

(a) Start

Serum

(b) After electrophoresis

(d) Precipitation arcs formed (c) Addition of antiserum

at equivalence

ANTISERUM

Fig. 3.10 Theoretical basis of immunoelectrophoresis. The antigen diffuses from a point source after the initial electrophoresis and interacts with the antiserum advancing on a plane front thus producing an arc of precipitation at equivalence.

Fig. 3.11 Immunoelectrophoresis of human serum. Sample (a): normal human serum showing normal IgG precipitation arc. Sample (b): serum of a patient with multiple myeloma. In this case the monoclonal protein is identified as IgG because of the ‘bump’ in the IgG precipitation arc towards the antiserum well. Antiserum in central trough: rabbit anti-human immunoglobulin. (Photograph of unstained preparation.)

98 C H A P T E R 3: Antibody interactions with antigens

TECHNICAL NOTE Although the relative distribution of the bands will depend on the batch of agar used and the initial electrophoresis distance, at the pH of the barbitone buffer (pH 8.2) the g-globulins are close to their isoelectric point and so would not migrate appreciably in the applied electric field. However, as mentioned earlier, the negative charge on the agar generates an electroendosmotic flow of water in the gel which sweeps the g-globulins towards the cathode. Often agarose is used as a supporting medium. This has less charge and so generates a lesser electroendosmotic flow.

3.7.3 Counterimmunoelectrophoresis

Gamma-globulins are exceptional in their cathodic migration; most other proteins move to the anode. The technique is similar to a one-dimensional Ouchterlony immunodiffusion but much faster as it is electrically driven, and more sensitive as all the antigen and antibody are driven towards each other. This property is used to advantage to cause antibody and antigen to migrate towards each other in the gel and form lines of precipitation.

MATERIALS AND EQUIPMENT As for agar electrophoresis plus Human serum albumin (HSA) Anti-HSA serum

METHOD

1 Prepare slide as for agar gel electrophoresis.

2 Punch two wells (5–10 mm apart).

3 Place anti-HSA in the anodal well and HSA in the cathodal well.

4 Run the slide in an electrophoresis tank as before.

5 After 10–15 min, examine for a line of precipitation. This technique lends itself to the rapid processing of many antisera or antigens.