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E. A. FREUNDT ET AL.
The optimal antigen dilution is that which gives a 50% haemolysis
endpoint with the highest dilution of antiserum. T o avoid or at least
minimise false positive reactions due to anticomplementary activity,
Mayer (1961) suggests that antigen when tested without antiserum should
not be anticomplementary at twice the concentration used in the test
proper. Quite frequently, two consecutive dilutions of antigen give the
same optimal endpoint. I n such a case, the rule quoted above may suggest
the use of the higher antigen dilution.
To select the working concentration of an antigen whose relationships
are unknown and for which no homologous antiserum is available, a
titration for anticomplementary activity should be carried out. T h e two
lowest dilutions that are not anticomplementary should then be used in the
test proper.
3. The test proper
The OSC of haemolysin, the dilution of complement containing 3
HC50 units and the optimal dilutions of the antigens have already been
determined.
The test is set up as described for the antigen titration in Section 2 (h):
antiserum is diluted in the plates from a primary dilution of 1 : 10 or 1 : 20 ;
one volume each of diluted antigen and complement are added to each
well and fixation is allowed to proceed at 4°C overnight; two volumes of
freshly washed and sensitised sheep erythrocytes are added to each well
and the test incubated for 60 min at 37°C.
A properly controlled test to identify an unknown mycoplasma should be
designed so that (i) each reference antiserum is titrated against the unknown antigen, its homologous antigen (positive control) and diluent
without antigen; (ii) where the corresponding preimmunization serum is
available, a similar titration is set up with serum dilutions between 1 : 10
and 1:80; (iii) each antigen is tested against 3, 1, 3 and 0 HC50 units of
complement (for anticomplementary activity).
I n our experience, a strain which reacts with a reference antiserum to
within 50 or 25% (i.e. one or two dilutions) of the homologous titre, can
be considered to belong to the same species as the homologous strain.
G. Double immunodiffusion
Two types of double immunodiffusion (DID) tests are described. T h e
first is a micro-test, which is carried out on microscope slides and is used to
screen sera and antigens for specific and non-specific activity, and for
determining the optimal antigen concentration. T h e second is on a slightly
larger scale and requires more materials, but has the advantage that
E. A. FREUNDT ET AL.
The optimal antigen dilution is that which gives a 50% haemolysis
endpoint with the highest dilution of antiserum. T o avoid or at least
minimise false positive reactions due to anticomplementary activity,
Mayer (1961) suggests that antigen when tested without antiserum should
not be anticomplementary at twice the concentration used in the test
proper. Quite frequently, two consecutive dilutions of antigen give the
same optimal endpoint. I n such a case, the rule quoted above may suggest
the use of the higher antigen dilution.
To select the working concentration of an antigen whose relationships
are unknown and for which no homologous antiserum is available, a
titration for anticomplementary activity should be carried out. T h e two
lowest dilutions that are not anticomplementary should then be used in the
test proper.
3. The test proper
The OSC of haemolysin, the dilution of complement containing 3
HC50 units and the optimal dilutions of the antigens have already been
determined.
The test is set up as described for the antigen titration in Section 2 (h):
antiserum is diluted in the plates from a primary dilution of 1 : 10 or 1 : 20 ;
one volume each of diluted antigen and complement are added to each
well and fixation is allowed to proceed at 4°C overnight; two volumes of
freshly washed and sensitised sheep erythrocytes are added to each well
and the test incubated for 60 min at 37°C.
A properly controlled test to identify an unknown mycoplasma should be
designed so that (i) each reference antiserum is titrated against the unknown antigen, its homologous antigen (positive control) and diluent
without antigen; (ii) where the corresponding preimmunization serum is
available, a similar titration is set up with serum dilutions between 1 : 10
and 1:80; (iii) each antigen is tested against 3, 1, 3 and 0 HC50 units of
complement (for anticomplementary activity).
I n our experience, a strain which reacts with a reference antiserum to
within 50 or 25% (i.e. one or two dilutions) of the homologous titre, can
be considered to belong to the same species as the homologous strain.
G. Double immunodiffusion
Two types of double immunodiffusion (DID) tests are described. T h e
first is a micro-test, which is carried out on microscope slides and is used to
screen sera and antigens for specific and non-specific activity, and for
determining the optimal antigen concentration. T h e second is on a slightly
larger scale and requires more materials, but has the advantage that