• The Complement Titration

Before the test, the basic solution of complement (1:10) is dispensed into a series of the test tubes in quantities varying from 0.05 to 0.5 ml, and then isotonic solution is added to each tube, bringing the volume of the fluid to 1.5 ml (see PRACTICAL WORK, item 3). Then the hemolytic system is added to them and the tubes are incubated for 30 min at 37^oC. After incubation the complement titer is measured. The titer of the complement is defined as its minimum concentration causing complete hemolysis in hemolytic system. To perform the test, one should take the working dose of the complement exceeding the titer by 20-30 per cent (the next tube that demonstrates hemolysis). The higher complement concentration is need (the abundance of the complement) to inhibit the anti-complement activity of the CF-test components.

• The Neutralization Reactions

The Neutralization Tests are antigen-antibody reactions in which the harmful effects of a bacterial exotoxin or a virus are eliminated by specific antibodies. Neutralization reactions are used both in diagnostic tests and in disease treatment, especially for diphtheria, botulism and tetanus. In such reactions a neutralizing antibodies called antitoxins combine with the exotoxin to neutralize it. Theses specific antibodies are produced by a host in response to exposure to the antigens of bacterial exotoxin or its corresponding toxoid (inactivated toxin) - see LESSON 15.

The neutralization tests are used in the diagnosis of toxemic bacterial and viral infections. Neutralization tests also allow to identify bacterial exotoxins. The antigenic type of toxin is identified by neutralization with specific antitoxin in mice (in vivo test, biological examination). For in vivo test exotoxin-containing specimen and several types specific antitoxic antisera are mixed in tubes respectively. Control tube contains the mixture of tested sample and isotonic solution. After 30 minutes incubation at room temperature 1 ml of mixture from each tube is injected intraperitoneally to mice. Mice injected with non-neutralized toxin die rapidly (a negative result of neutralization test). If toxin-antitoxin neutralization takes place (with the corresponding type of antitoxin), mouse will survive (positive result).

A more frequent form of neutralization test is a skin test such as the Schick test, which determines the status of a person's immunity to diphtheria. In this test, a small amount of diphtheria exotoxin is inoculated into the person's skin. If there is sufficient serum antitoxin to neutralize the exotoxin, there is no visible reaction, indicating that the person would be immune to diphtheria (positive test of neutralization). If antitoxin is insufficient, the exotoxin damages the tissues at the site of injection and produces a swollen, tender, reddish area that turns brown in four to five days (negative result).

Practical work

1. The Bacteriolysis Test. In carrying out the bacteriolysis reaction, the immune serum (patient’s serum) is heated for 30 min at 56^oC to inactivate the complement present in it. Suspension of microorganisms is prepared from their 24-hour culture (1 milliard (billion) CFU/ml). This suspension is added to the serum diluted 1:10 (or 1:20, 1:40, etc.). To the serum control tube no complement is added. In the complement control the immune serum is replaced with isotonic solution.

The bacteriolysis test may be performed both in vitro and in vivo. To perform the in vitro test after incubation at 37 ^OC for 2 h., the material of each tube is streaked with inoculating loop on MPA. After incubation at 37^oC for 24 h the bacterial growth/no growth on MPA is checked. If the serum to be tested contained bacteriolysins, no growth of bacterial colonies is seen. And the growth in the control tubes should be observed.

For in vivo test, the contents of all tubes after first step of examination (mixing the components and their incubation at 37^oC for 2 h.) are injected intraperitoneally into the mice (or guinea pigs). The peritoneal fluid is then examined microscopically 10 min, 20 min, and 1 h. after injection.

The Procedure of the Bacteriolysis Test. This test is performed in four tubes. The test tube contains the immune antiserum, suspension of the microorganisms, complement and isotonic solution. The control tubes do not contain either one component: the immune antiserum (2), the complement (3), or both immune antiserum and the complement (4) (see Table 13-1).

Table 13-1

The Scheme of the Bacteriolysis Test

Ingredients	Test 1	Control 2	Control 3	Control 4
Immune antiserum (ml)	0.2	--	0.2	--
Bacterial suspension (ml)	0.1	0.1	0.1	0.1
Complement (ml)	0.3	0.3	--	--
Isotonic solution/MPB (ml)	1.4	1.6	1.7	1.9
Total volume (ml)	2.0	2.0	2.0	2.0
Incubation at 37oC for 2 h.
Results (transparency of the fluid)
Inoculation of MPA, incubation at 37oC for 24 h.
Results (growth/no growth):
Conclusion: the bacteriolysis test is _______________________(positive/negative).

To estimate the results tubes should be checked for the transparency (growth/no growth). The materials of each four tubes are then subcultured on MPA divided on four parts. After 24 h. incubation at 37^oC results are examined. If the bacteriolysis test is positive, the fluid in the tested tube is transparent and no growth appears in the first sector of MPA. Control tubes, and negative test show not-transparent liquids and growth on MPA.

2. The Immune Hemolysis Test. The immune hemolysis test is employed for the complement titration and as an indicator system in the complement-fixation test. Thus, the hemolysis reaction is not used independently.

The demonstration of the Immune Hemolysis Test show the immune mechanism of hemolysis to the contrast of other types of hemolysis (e.g., due to physical or chemical damage of red blood cells).

Table 13-2

The Scheme of the Immune Hemolysis Test

Ingredients	Test 1	Control 2	Control 3	Control 4	Control 5
Rabbit's hemolytic antiserum (ml)	0.5	0.5	--	0.5	--
Sheep erythrocytes suspension (ml)	0.5	0.5	0.5	--	0.5
Complement in dilution 1:10 (ml)	0.5	--	0.5	0.5	--
Dog's erythrocytes suspension (ml)	--	--	--	0.5	--
Isotonic solution (ml)	--	0.5	0.5	--	1.0
Total volume of the mixture (ml)	1.5	1.5	1.5	1.5	1.5
Incubation at 37oC for 1h.
Results (hemolysis/no hemolysis):
Conclusion: the immune hemolysis test is _____________________.

This test is carried out in the row of five tubes, and only one of them is the test tube (see Table 13-2). If the reaction is positive, a complete hemolysis occurs, and the fluid in the tube is intensely pink ( «varnished blood»). The positive reaction is denoted with plus. In negative test (-), there is no hemolysis and the fluid remains red and non-transparent.

3. The Procedure of the Complement Titration. This test is performed in the row of ten tubes.

1. Take ten grease-free non-sterile tubes.

2. Dispense various increasing amounts of basic solution of complement (initial dilution 1:10) from 0.15 to 0.5 ml into the tubes (as it is shown in Table 13-3).

3. Add the isotonic solution into each tube to adjust the volume of the fluid to 1.5 ml. Thus, you have obtained the various dilutions of the complement: the first tube contains the lowest complement concentration, and the last one (#8) contains its highest amount.

4. Flood 0.5 ml of complement and 1.5 ml of isotonic solution into the first control tube (#9). This is the complement control.

5. Pour 1.5 ml of isotonic sodium chloride solution into the next control tube (#10). This is the hemolytic system control.

6. Add hemolytic system into each tube (see the Table).

7. Incubate the tubes at 37^oC for 30 min.

8. Read the results. Denote the presence of hemolysis with (+). Point out negative results with (-).

Table 13-3

The Scheme of the Complement Titration Test

Ingredients	1	2	3	4	5	6	7	8	Control 9	Control 10
Complement in 1:10 dilution (ml)	0.15	0.2	0.25	0.3	0.35	0.4	0.45	0.5	0.5	--
Isotonic solution (ml)	1.35	1.3	1.25	1.2	1.15	1.1	1.05	1.0	1.5	1.5
Hemolytic system (ml)	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	0.5	1.0
Incubation at 37oC for 30 min.
Results (hemolysis/no hemolysis:
Conclusion: the titer of complement is _______ ml; the working dose of complement is _________ ml.

One should begin to estimate the test results with the controls. The hemolytic system control must be checked for the absence of spontaneous hemolysis. A complete hemolysis must be observed in the complement control. To determine the complement titer one should note the first tube in the row where the hemolysis appears. This is the titer of the complement (it is measured in ml). The next tube with hemolysis is the working dose of the complement.

4. The Procedure of the Complement Fixation Test to detect of unknown antibodies in the patient’s serum. The performance of CFT requires at least two controls (serum control and antigen control). The main steps of procedure are as follows (see Table 13-4):

1. Flood 0.5 ml of the patient's serum in the appropriate dilution into the test tube and the same volume into the serum control tube.

2. Add 0.5 ml of the antigen (diagnosticum) and 0.5 ml of the complement to the first tube.

3. Dispense 0.5 ml of complement and 0.5 ml of isotonic solution (instead of the antigen) in the second tube (serum control).

4. Pour the same volumes (0.5 ml) of antigen, complement and isotonic solution into the third tube (antigen control).

5. Place the tubes into the 37^oC incubator. Allow them to stand for 1 h.

6. Take the tubes from the incubator and add 1.0 ml of hemolytic system to each tube.

7. Reincubate the tubes at 37^oC for 1 h.

8. Read the results beginning with the controls. Note the presence of hemolysis in the control tubes. If the CF-test is positive, no hemolysis is registered in the test tube. If the test is negative, the complete hemolysis occurs in the test tube. Register the results in Table 13-4.

Table 13-4

The Scheme of Complement Fixation Test.

Ingredients	Test tube 1	Serum control 2	Antigen control 3
Patient’s serum in dilution 1:25 (ml)	0.5	0.5	--
Antigen (diagnosticum) (ml)	0.5	--	0.5
Complement (working dose) (ml)	0.5	0.5	0.5
Isotonic solution (ml)	--	0.5	0.5
Incubation at 37oC for 1 h.
Hemolytic system (ml)	1.0	1.0	1.0
Incubation at 37oC for 1 h.
Results: (hemolysis/no hemolysis)
Results: (hemolysis/no hemolysis)
Conclusion: the CFT is ___________________(positive/negative). The patient’s serum contains (does not contain) specific antibodies.