Immune Body

Immune body or amboceptor is the name given to a substance present in the serum of an infected animal that has successfully resisted inoculation with some particular micro-organism, and which possesses the power of linking the complement normally present in the serum to bacteria of the species used as antigen in such a manner that the micro-organisms are rendered innocuous, and ultimately destroyed. The presence of the immune body in the serum can be demonstrated in vitro by the reaction elaborated by Bordet and Gengou, known as the complement fixation test, the existence or the absence of the phenomenon of complement fixation being rendered obvious macroscopically by the absence or presence of hæmolysis on the subsequent addition of "sensitised" red blood corpuscles, (e. g., a mixture of crythrocyte solution and the appropriate hæmolysin—two of the three essentials in the hæmolytic system, vide page 326).

Apparatus Required:

Sterile pipettes 1 c.c., (graduated in tenths).

16 × 2 cm. test-tubes.

9 × 1 cm. test-tubes.

Test-tube racks for each size of test-tube.

Reagents Required:

Normal saline solution.

Erythrocyte solution (human red cells, page 329) = E.

Hæmolytic serum (for human cells) = H.S.

Complement (fresh guinea-pig serum) = C.

Specific serum from inoculated animal, inactivated = S.S.

Control pooled serum from normal animals of same species, Inactivated = P.S.

Antigen (cultivation upon solid medium of the organism (e. g., B. typhosus) which has already served as antigen in the inoculation of the experimental animal) = A.

To prepare the antigen for use, emulsify the whole of the bacterial growth in 5 c.c. normal saline solution.

Shake the emulsion in a test-tube with some sterilised glass beads to ensure a homogenous emulsion, and sterilise by heating to 60° C. in a water-bath for one hour.

Method.—

1. Take five small test-tubes, and number them 1 to 5 with a grease pencil.

2. Into tubes Nos. 1, 3, 4 and 5 pipette 0.1 c.c. of complement.

3. Into tubes Nos. 1 and 2 pipette 0.2 c.c. of the serum to be tested.

4. Into tube No. 4 pipette 0.2 c.c. of control serum.

5. Into tubes Nos. 1, 2, 3 and 4 pipette 1 c.c. of the bacterial emulsion which forms the antigen.

6. Place the whole set of tubes in the incubator at 37° C. for a period of one hour.

7. Remove the tubes from the incubator and pipette 1 c.c. erythrocyte solution and 4 minimal hæmolytic doses of the corresponding hæmolysin into each tube.

8. Mix thoroughly and return the tubes to the incubator at 37° C. for further period of one hour.

9. At the expiration of that time transfer the tubes to the ice chest, and allow them to stand for three hours.

10. Examine the tubes.

Tubes 3, 4 and 5 should show complete hæmolysis; tube 2 should give no evidence whatever of hæmolysis.

These tubes form the controls to the first tube, which contains the serum to be tested.

In tube No. 1 the absence of hæmolysis would indicate the presence in the serum of the inoculated animal of a specific antibody to the micro-organism used in the inoculations; since it shows that the complement has been bound by the immune body to the bacterial antigen, and none has been left free to enter into the hæmolytic system; on the other hand the presence of hæmolysis would show that no appreciable amount of antibody has yet been formed in response to the inoculations. In other words, there is an absence of infection, since the complement remained unfixed at the time of the addition of the erythrocyte solution and hæmolytic serum, and was ready to combine with those reagents to complete the hæmolytic system.

The method may be shown diagramatically as under using the symbols already indicated

Note.—It is sometimes more convenient to sensitise the erythrocytes just before they are needed. This is done forty-five minutes after the experiment has been started (page 394, step 6), that is to say, before the completion of the first period of incubation, thus:

1. Measure out into a sterile test-tube (or flask) five c.c. of erythrocyte solution.

2. Measure out twenty minimal hæmolytic doses of hæmolysin, add to the erythrocyte solution on the test-tube.

3. Allow the erythrocyte and hæmolysin to remain in contact for fifteen minutes at room temperature. The red cells are then sensitised and ready for use.

4. When the tubes are removed from the incubator at the end of the first hour (i. e., step 7) add 1 c.c. sensitised red cells to each tube by means of a graduated pipette.

5. Mix thoroughly, return the tubes to the incubator at 37°C. and complete the experiment as previously described (steps 8 onward).