Total haemolytic complement
7.5 Total haemolytic complement
Lysis of antibody-coated erythrocytes has long been used as a means of estimating the comple- ment activity of a serum. As complement is added to antibody-coated erythrocytes an increas- ing proportion of the cells are lysed as shown in Fig. 7.3. As the curve approaches 100% lysis asymptotically, it is difficult to determine the total lytic unit of complement (CH 100 ) and so one
normally defines the 50% lysis point (CH 50 ). The von Krogh equation for the sigmoid dose–response curve of complement-mediated cytolysis was derived empirically and, in its basic form, may be written as:
1 / ⎧ n y ⎫ x= k ⎨
⎬ ⎩ 100 − y ⎭ where x is amount of complement (ml of undiluted serum), y is proportion of cells lysed, k is 50%
unit of complement and n is a constant.
C H A P T E R 7: Phagocytosis, complement and antibody-dependent cytotoxicity
Fig. 7.3 Lysis of sheep erythrocytes
Lysis (%)
(SRBC), sensitized by horse anti- SRBC, in the presence of human
complement. The curve of complement-mediated lysis approaches the 100% lysis value asymptotically and so accurate determinations of serum
0 1 5 10 complement levels are made on the
Volume of complement added (arbitrary units) 50% lysis point as shown in the graph.
The CH 50 unit is determined under standardized conditions which depend upon: (a) erythrocyte and antibody concentration; (b) buffering conditions of the medium; and (c) temperature.
Hence, the definition of the CH 50 unit is arbitrary and depends on the conditions used. The assay may be performed in tubes without reference to a standard, or in agar with reference to a standard serum.
7.5.1 Standardization of erythrocytes
MATERIALS AND EQUIPMENT Barbitone-buffered saline for complement tests (this contains essential calcium and magnesium ions)
Sheep erythrocytes (SRBC) in Alsever’s solution 0.04% ammonia solution Serum: this should be either fresh, or guinea-pig serum preserved specially for complement fixation
assays Horse haemolytic serum (source of anti-erythrocyte antibody) Spectrophotometer
METHOD
1 Dilute the barbitone-buffered saline to working strength. Check for fungal or bacterial contamination, as these are anticomplementary.
2 Wash 4 ml of the erythrocyte suspension (supplied at ≈ 25% v/v in Alsever’s solution) three times in barbitone-buffered saline (200 g for 3 min).
3 Resuspend the washed erythrocytes in 15 ml of barbitone-buffered saline (use a measuring cylinder).
4 Mix 1 ml of erythrocytes with 25 ml of ammonia solution to lyse the cells and read the absorbance at 541 nm. For a 6% SRBC suspension, in a 1-cm cuvette, the absorbance should be 0.48–0.50. Adjust the suspension as required.
Continued on p. 218
7.5TOTAL HAEMOLYTIC COMPLEMENT
5 Mix 15 ml barbitone-buffered saline, 0.1 ml of horse haemolytic serum and 15 ml of 6% SRBC. Strictly, the anti-erythrocyte serum should be titred until the highest dilution still giving full complement fixation is reached; however, for most purposes it is sufficient to use a 1 : 150 dilution.
6 Incubate at 37°C for 15 min. This method is for the preparation of 30 ml of 3% v/v sheep erythrocytes. Use the sensitized cells within 24 h.
7.5.2 Estimation of CH 50 tube assay
METHOD
1 Set up the tubes as in the Protocol and remember to use fresh or specially preserved serum as the complement source.
2 Incubate at 37°C for 60 min.
3 Place the tubes on ice and add 2 ml of buffer to each tube.
4 Centrifuge at 200 g for 10 min at 4°C.
5 Remove a sample of each supernatant (tubes 1–7) and read their absorbance at 541 nm.
Protocol.
Tube numbers
1.10 1.05 1.00 0.90 0.80 l.20 1.20 ml ammonia solution Guinea-pig serum (ml) initial dil. 1 : 30
Buffer (ml)
0.10 0.15 0.20 0.30 0.40 0.00 Sensitized erythrocytes (ml) suspension 0.3⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→
Calculation of results
1 Assuming that tube 7 represents total lysis, calculate the percentage lysis for each tube.
2 Plot the percentage lysis against the complement concentration (ml of undiluted serum). This will yield a sigmoid curve as in Fig. 7.3.
This dose–response curve follows the von Krogh equation given earlier. However, this equation may be logarithmically transformed so that the data fall on a straight line:
logx = logk + 1/n log y
100 y
where terms are defined as previously.
3 Plot log x against log[y/(100 – y)] for each dilution of complement used. The straight line has
a slope of 1/n (the exact value depends on experimental conditions, but it should be within 20% of 0.2). The abscissa intercept of the line, where log[y/(100 – y)] = 0, is the log dilution
C H A P T E R 7: Phagocytosis, complement and antibody-dependent cytotoxicity C H A P T E R 7: Phagocytosis, complement and antibody-dependent cytotoxicity
of CH 50 units/ml of serum. TECHNICAL NOTES
• Complement components are highly labile and so fresh serum must be prepared by clotting the
blood at 4°C. Preserved guinea-pig serum is available commercially, in which the complement components are stabilized by lyophilization of serum in a hypertonic salt solution.
• The whole assay may be made more sensitive, and use less reagents, if the red cells are radio- labelled with 51 Cr for the radioisotopic variant of this assay. • Instead of erythrocytes, algal cells from Euglena gracilis (Stefanski & Ruppel 1991) or fluor-
escently labelled liposomes (Masaki et al. 1989) may be used to increase sensitivity and reproducibility.
• The assay may be modified for use on microtitre plates.
7.5.3 Estimation of CH 100 by assay in agar
This simple assay, which is analogous to single radial immunodiffusion, may be used on a routine basis with reference to a standard serum. Antibody-sensitized red cells are incorporated into molten agarose and the mixture allowed to set. Wells are cut in the agarose and filled with either the sera under test or dilutions of a standard serum. The complement diffuses into the agarose and reacts with and binds to the antibody-coated red cells. Circles of lysis appear, the size of which depend upon the complement content of the serum.
MATERIALS AND EQUIPMENT All reagents should be made up in barbitone buffer for complement fixation Sensitized red cells (made up to 10% v/v) Agarose, 2% w/v in barbitone buffer Glass plates (microscope slides are suitable for a small number of estimations) Gel cutter
METHOD
1 Warm 1.5 ml of the barbitone buffer to 56°C in a water bath.
2 Cool 1.2 ml of molten 2% agarose to 56°C and add to the barbitone buffer.
3 Mix and cool to 45°C in a water bath.
4 Add 0.2 ml of sensitized red-cell suspension and mix gently.
5 Place the glass plate on a level surface; use a spirit level to check.
6 Pour the mixture quickly onto the plate to form a smooth, even surface.
7 When set, place the plate in a box containing moist filter paper and chill to 4°C for a few minutes to harden the agarose.
8 Cut two rows of five wells, approximately 3 mm across using an Ouchterlony gel cutter and remove the agarose plugs with a Pasteur pipette attached to a Venturi pump.
9 Dispense 8 µl samples of the sera under test into separate wells. Similarly, add four doubling dilutions of a standard serum to a series of wells. (For accurate research studies, a larger plate can be used to permit replicate determinations.)
10 Incubate the plate in a moist box overnight at 4°C.
Continued on p. 220
7.5TOTAL HAEMOLYTIC COMPLEMENT
11 Warm the plate, still in the box, to 37°C for 2 h, to allow cell lysis to occur.
12 Measure two diameters at right angles across each well and calculate their mean.
13 Plot the value of the areas (pr 2 ) of the standard serum dilutions (linear scale) against the log dilution. Determine the concentration of the unknown sera as a percentage of the standard by extrapolation from the standard curve.
TECHNICAL NOTE This technique is eminently suitable for detecting complement deficiencies both in total and in individual components of complement. Qualitative assay reagents may be prepared in which just one component is missing from the lytic pathway of complement. These reagents are incorporated in agarose and will only lyse the indicator erythrocytes if the missing component is present in the test serum added to the well.