414
E. A. FREUNDT ET AL.
antibodies, but adapted to a microtitre system using 0.025 ml as unit
volume. In the test proper,. the primary reaction mixture comprises one
volume each of antiserum, antigen and complement, fixation of the latter
being allowed to proceed overnight at 4°C. The indicator system consists
of two volumes of sheep erythrocytes optimally sensitised with haemolysin.
The endpoint of all titrations is recorded as that dilution which shows 50%
haemolysis of the indicator system. The procedures to be described
minimise cross-reactions between different species.
1. EquzPment and cleaning procedures
The microtitre system used is essentially the same as that described for
the MI test. Permanent lucite plates with U-shaped wells are satisfactory
if washed by the procedure of Sever (1962). Plates are soaked in detergent
(Haemosol, Mainecke and Co., Inc., Baltimore, U.S.A. : available from
Alfred Cox, Surgical Ltd, Coulsdon, Surrey, England) at 40-50°C for
30 min, rinsed in warm tap water, then in deionised water and finally dried
at 3740°C. During each wash or rinse operation, the plates are scrubbed
with a hand brush, filled and shaken empty several times.
2. Reagents
These include diluent, sheep erythrocytes, complement, haemolysin,
antigen and antiserum.
(a) Diluent. A barbitone buffer containing Ca2+ and Mg2+ is used throughout. This is most conveniently prepared from Oxoid Barbitone C F T
diluent tablets (Code BR16, Oxoid Ltd, London S.E.l). Water for preparing the diluent should be freshly glass-distilled or, if more crudely
distilled, also deionised. Alternatively, barbitone buffer may be prepared
from its constituent chemicals as described by Cruickshank et al. (1975).
(b) Sheep erythrocytes preserved in Alsever's solution. Satisfactory products
are available from commercial sources (Wellcome Reagents Ltd) and can
be stored at 4°C. Alsever's solution and buffy coat are aspirated after an
initial centrifugation, and the red cells washed three times in 0.85%
saline. The packed erythrocytes are then resuspended in diluent to a
concentration of 2%. The addition of an equal volume of the appropriate
dilution of haemolysin to this suspension means that the final concentration
of red cells is 1%. This is suitable for the microtitre system because it
gives cell buttons and/or coloured supernatants which are easily interpreted
in terms of percentage haemolysis.
(c) Complement. Pooled guinea pig serum preserved by Richardson's
method (Cruickshank et al., 1975) and stored at 4"C, or frozen without
preservative and stored at - 70°C, is the source of complement. Preserved
E. A. FREUNDT ET AL.
antibodies, but adapted to a microtitre system using 0.025 ml as unit
volume. In the test proper,. the primary reaction mixture comprises one
volume each of antiserum, antigen and complement, fixation of the latter
being allowed to proceed overnight at 4°C. The indicator system consists
of two volumes of sheep erythrocytes optimally sensitised with haemolysin.
The endpoint of all titrations is recorded as that dilution which shows 50%
haemolysis of the indicator system. The procedures to be described
minimise cross-reactions between different species.
1. EquzPment and cleaning procedures
The microtitre system used is essentially the same as that described for
the MI test. Permanent lucite plates with U-shaped wells are satisfactory
if washed by the procedure of Sever (1962). Plates are soaked in detergent
(Haemosol, Mainecke and Co., Inc., Baltimore, U.S.A. : available from
Alfred Cox, Surgical Ltd, Coulsdon, Surrey, England) at 40-50°C for
30 min, rinsed in warm tap water, then in deionised water and finally dried
at 3740°C. During each wash or rinse operation, the plates are scrubbed
with a hand brush, filled and shaken empty several times.
2. Reagents
These include diluent, sheep erythrocytes, complement, haemolysin,
antigen and antiserum.
(a) Diluent. A barbitone buffer containing Ca2+ and Mg2+ is used throughout. This is most conveniently prepared from Oxoid Barbitone C F T
diluent tablets (Code BR16, Oxoid Ltd, London S.E.l). Water for preparing the diluent should be freshly glass-distilled or, if more crudely
distilled, also deionised. Alternatively, barbitone buffer may be prepared
from its constituent chemicals as described by Cruickshank et al. (1975).
(b) Sheep erythrocytes preserved in Alsever's solution. Satisfactory products
are available from commercial sources (Wellcome Reagents Ltd) and can
be stored at 4°C. Alsever's solution and buffy coat are aspirated after an
initial centrifugation, and the red cells washed three times in 0.85%
saline. The packed erythrocytes are then resuspended in diluent to a
concentration of 2%. The addition of an equal volume of the appropriate
dilution of haemolysin to this suspension means that the final concentration
of red cells is 1%. This is suitable for the microtitre system because it
gives cell buttons and/or coloured supernatants which are easily interpreted
in terms of percentage haemolysis.
(c) Complement. Pooled guinea pig serum preserved by Richardson's
method (Cruickshank et al., 1975) and stored at 4"C, or frozen without
preservative and stored at - 70°C, is the source of complement. Preserved