IX. IDENTIFICATION OF MYCOPLASMAS
419
3, 2, 1, 4 and 0 HC50 units of Complement. One drop each of diluent and
the appropriate complement dilution completes the primary reaction
mixture. Two rows in which diluent is substituted for antigen are included
as controls.
Serum to be tested is first diluted 1:lO in diluent and inactivated at
56°C for 30 min. From this, two-fold dilutions up to 1 : 80 are made in the
plate as described for the M I test. One drop each of diluent and 3 HC50
complement is added to each well. T o establish that there is no procomplementary activity, a second row of serum dilutions in which complement is replaced by diluent may be set up.
Next morning, a 2% suspension of washed sheep cells is sensitised for
60 min at 37°C with an equal volume of haemolysin at the OSC. Two
drops are added to each well and the tests completed as described for the
haemolysin-complement titration.
Antigen is only acceptable at a given dilution if it gives the same reaction
as the control rows without antigen. Since by definition 1 HC50 of complement lyses 50% of the red cells, the control rows should give the
reactions shown in Table VI(a). I n this example, the antigen is not anticomplementary at 1 : 8 and above. Dilutions which are not anticomplementary are then titrated to determine the optimal antigen concentration.
Antisera or preimmunisation sera which are anticomplementary can be
diluted to 1 : 10 with a 1 : 10 dilution of complement and reacted for 24-48 h
at 4°C. After inactivation at 56°C for 30 min, sera are retested, but the
second row from which complement has been omitted must be included to
establish that the complement used for absorption has been inactivated.
Absorbed inactivated sera can be stored as dilutions for short periods at or
below -30°C. The guinea pig serum used as the source of complement
could contain antibodies which react with mycoplasma antigens, although
such antibodies have not been detected in the batches of pooled serum
which we have used.
(h) Titration of antigen. It has been shown that there is an equivalence zone
with respect to concentrations of antigen and antibody, and that fixation of
complement is optimal at or near this zone. Inhibition of complement
fixation occurs as the quantity of antigen is increased beyond that optimum
(Mayer, 1961). T o determine the optimal antigen dilution, therefore, a
series of antigen dilutions is titrated against dilutions of the homologous
antiserum or an antiserum against a strain of the same species. For this
test, antiserum is diluted in the plates, antigen dilutions are prepared in
tubes and one drop added to the wells. One drop of 3 HC50 complement
completes the primary reaction mixture. The test is completed as described
in the preceding Section.
419
3, 2, 1, 4 and 0 HC50 units of Complement. One drop each of diluent and
the appropriate complement dilution completes the primary reaction
mixture. Two rows in which diluent is substituted for antigen are included
as controls.
Serum to be tested is first diluted 1:lO in diluent and inactivated at
56°C for 30 min. From this, two-fold dilutions up to 1 : 80 are made in the
plate as described for the M I test. One drop each of diluent and 3 HC50
complement is added to each well. T o establish that there is no procomplementary activity, a second row of serum dilutions in which complement is replaced by diluent may be set up.
Next morning, a 2% suspension of washed sheep cells is sensitised for
60 min at 37°C with an equal volume of haemolysin at the OSC. Two
drops are added to each well and the tests completed as described for the
haemolysin-complement titration.
Antigen is only acceptable at a given dilution if it gives the same reaction
as the control rows without antigen. Since by definition 1 HC50 of complement lyses 50% of the red cells, the control rows should give the
reactions shown in Table VI(a). I n this example, the antigen is not anticomplementary at 1 : 8 and above. Dilutions which are not anticomplementary are then titrated to determine the optimal antigen concentration.
Antisera or preimmunisation sera which are anticomplementary can be
diluted to 1 : 10 with a 1 : 10 dilution of complement and reacted for 24-48 h
at 4°C. After inactivation at 56°C for 30 min, sera are retested, but the
second row from which complement has been omitted must be included to
establish that the complement used for absorption has been inactivated.
Absorbed inactivated sera can be stored as dilutions for short periods at or
below -30°C. The guinea pig serum used as the source of complement
could contain antibodies which react with mycoplasma antigens, although
such antibodies have not been detected in the batches of pooled serum
which we have used.
(h) Titration of antigen. It has been shown that there is an equivalence zone
with respect to concentrations of antigen and antibody, and that fixation of
complement is optimal at or near this zone. Inhibition of complement
fixation occurs as the quantity of antigen is increased beyond that optimum
(Mayer, 1961). T o determine the optimal antigen dilution, therefore, a
series of antigen dilutions is titrated against dilutions of the homologous
antiserum or an antiserum against a strain of the same species. For this
test, antiserum is diluted in the plates, antigen dilutions are prepared in
tubes and one drop added to the wells. One drop of 3 HC50 complement
completes the primary reaction mixture. The test is completed as described
in the preceding Section.