422
E. A. FREUNDT ET AL.
Amidoschwarz (Naphthalene black) 10B
or
6% acetic acid
45 ml
0.1 M sodium acetate
45 ml
Glycerol
10 ml
Ponceau fuchsin
0.1 g
The decolourising solution consists of:
Glycerol
15 ml
Glacial acetic acid
2 ml
Distilled or deionised water
83 ml
(c) Antisera. For D I D tests, rabbit antisera have advantages over horse
antisera, because the antigen-antibody precipitates obtained with the
former are much less soluble in the presence of excess antigen or antibody.
Also, rabbit serum is less likely to give rise to artifacts due to changes in
temperature (Crowle, 1961). Antisera for D I D tests should be free from
antibodies to foreign serum proteins (Section V. A). Moreover, satisfactory
precipitating sera usually require the use of Freund's complete or incomplete adjuvant in their production. With mycoplasma antigens, more lines
are given by adjuvant-produced antisera than by antisera produced by
intravenous inoculation. However, the additional lines given by adjuvantproduced sera are mainly due to cytoplasmic antigens (Lemcke, 1973).
Tests should be set up to make sure that neither preimmunisation sera
nor antisera react with any of the medium constituents used to grow the
antigens. The sediment obtained after centrifugation of the medium has
often been used, but media supplemented with bovine serum fraction
(Difco) are virtually clear and give very little deposit. It may therefore be
more convenient to test the undiluted sera against the complete medium
and those niedium constituents which are most likely to be antigenic. T h e
test may be set up with serum in the central well and medium constituents
or complete medium alternating with the homologous antigen in the
peripheral wells. Such a test will also indicate whether any of the lines
given by the antigen are due to adsorbed medium constituents, since the
reaction of an adsorbed component will be enhanced by the greater
concentration of that component in the adjacent well. I n our experience,
cross-reactions between antisera from rabbits immunised with mycoplasmas grown in rabbit serum broth and components adsorbed to antigens
grown in serum fraction media have not been a problem. If such reactions
are detected, the reacting antibody can be absorbed from the antiserum
by including the medium component or the complete medium in the geldiffusion agar at a concentration of lo'-); (v/v) (Hollingdale and Lemcke,
E. A. FREUNDT ET AL.
Amidoschwarz (Naphthalene black) 10B
or
6% acetic acid
45 ml
0.1 M sodium acetate
45 ml
Glycerol
10 ml
Ponceau fuchsin
0.1 g
The decolourising solution consists of:
Glycerol
15 ml
Glacial acetic acid
2 ml
Distilled or deionised water
83 ml
(c) Antisera. For D I D tests, rabbit antisera have advantages over horse
antisera, because the antigen-antibody precipitates obtained with the
former are much less soluble in the presence of excess antigen or antibody.
Also, rabbit serum is less likely to give rise to artifacts due to changes in
temperature (Crowle, 1961). Antisera for D I D tests should be free from
antibodies to foreign serum proteins (Section V. A). Moreover, satisfactory
precipitating sera usually require the use of Freund's complete or incomplete adjuvant in their production. With mycoplasma antigens, more lines
are given by adjuvant-produced antisera than by antisera produced by
intravenous inoculation. However, the additional lines given by adjuvantproduced sera are mainly due to cytoplasmic antigens (Lemcke, 1973).
Tests should be set up to make sure that neither preimmunisation sera
nor antisera react with any of the medium constituents used to grow the
antigens. The sediment obtained after centrifugation of the medium has
often been used, but media supplemented with bovine serum fraction
(Difco) are virtually clear and give very little deposit. It may therefore be
more convenient to test the undiluted sera against the complete medium
and those niedium constituents which are most likely to be antigenic. T h e
test may be set up with serum in the central well and medium constituents
or complete medium alternating with the homologous antigen in the
peripheral wells. Such a test will also indicate whether any of the lines
given by the antigen are due to adsorbed medium constituents, since the
reaction of an adsorbed component will be enhanced by the greater
concentration of that component in the adjacent well. I n our experience,
cross-reactions between antisera from rabbits immunised with mycoplasmas grown in rabbit serum broth and components adsorbed to antigens
grown in serum fraction media have not been a problem. If such reactions
are detected, the reacting antibody can be absorbed from the antiserum
by including the medium component or the complete medium in the geldiffusion agar at a concentration of lo'-); (v/v) (Hollingdale and Lemcke,