294
M. A . GERENCSER
fluorescein isothiocyanate and used to stain Actinonlyces grown in AB or
PDT, the results were uniformly negative. Since antigenic components of
the medium do not seem to interfere with FA results, media giving faster
growth and higher cell yields than PDT are now used routinely for growing
immunising antigens. These media, Trypticase Soy Broth, Brain-Heart
Infusion Broth, and Actinomyces Broth, have also been used for this
purpose by other workers (Lambert et al., 1967; Bellack and Jordan, 1972;
Holmberg and Forsum, 1973).
2. Immunisation schedules
Rabbits are bled prior to immunisation, the serum collected and stored
frozen. Two immunisation schedules used in our laboratory are shown in
Table IV. Schedule 1 is the one now used regularly and generally gives
good results although an occasional rabbit does not respond. Similar
schedules have been used by other workers (Lambert et al., 1967).
The second schedule which has also been used to produce good antiserum
is shown for comparison since it employs an adjuvant and subcutaneous
injections as well as intravenous injections.
While both of these schedules and others reported in the literature
(Bellack and Jordan, 1972; Bowden et al., 1976) yield usable antiserum,
there is no evidence to indicate that any one injection schedule is optimal.
This is illustrated by the variability in antiserum produced in response to
the same lot of antigen given by the same injection schedule. Some of this
is undoubtedly due to variations in individual animal response, but this is
not the sole explanation. Antiserum differences are most noticeable when
used in immunodiffusion or similar tests, but FA conjugates are also
affected. This problem has been discussed by Bowden and Hardie (1973),
Landfried (1966, 1972) and by Holmberg et al. (1975a).
3. Evaluation of the antiserum
Antibody titres, either during the course of the immunisation or at the
completion of the schedule, can be determined by the agglutination,
precipitin or by indirect FA tests. Unfortunately none of these tests
correlates well with subsequent FA titres of fluorescein isothiocyanate
conjugates made from the serum. They do, however, indicate that an
antibody response has occurred and serve to weed out animals with very
poor titres. I n general, a serum which has a high titre in agglutination
tests or by indirect FA will give a satisfactory conjugate. As a rough guide,
we have found that antiserum with an indirect FA titre of 1 : 500 or greater,
usually yields a usable conjugate. It should be pointed out that there is no
direct correlation between the indirect titre of the whole serum and the
titre of the conjugate.
M. A . GERENCSER
fluorescein isothiocyanate and used to stain Actinonlyces grown in AB or
PDT, the results were uniformly negative. Since antigenic components of
the medium do not seem to interfere with FA results, media giving faster
growth and higher cell yields than PDT are now used routinely for growing
immunising antigens. These media, Trypticase Soy Broth, Brain-Heart
Infusion Broth, and Actinomyces Broth, have also been used for this
purpose by other workers (Lambert et al., 1967; Bellack and Jordan, 1972;
Holmberg and Forsum, 1973).
2. Immunisation schedules
Rabbits are bled prior to immunisation, the serum collected and stored
frozen. Two immunisation schedules used in our laboratory are shown in
Table IV. Schedule 1 is the one now used regularly and generally gives
good results although an occasional rabbit does not respond. Similar
schedules have been used by other workers (Lambert et al., 1967).
The second schedule which has also been used to produce good antiserum
is shown for comparison since it employs an adjuvant and subcutaneous
injections as well as intravenous injections.
While both of these schedules and others reported in the literature
(Bellack and Jordan, 1972; Bowden et al., 1976) yield usable antiserum,
there is no evidence to indicate that any one injection schedule is optimal.
This is illustrated by the variability in antiserum produced in response to
the same lot of antigen given by the same injection schedule. Some of this
is undoubtedly due to variations in individual animal response, but this is
not the sole explanation. Antiserum differences are most noticeable when
used in immunodiffusion or similar tests, but FA conjugates are also
affected. This problem has been discussed by Bowden and Hardie (1973),
Landfried (1966, 1972) and by Holmberg et al. (1975a).
3. Evaluation of the antiserum
Antibody titres, either during the course of the immunisation or at the
completion of the schedule, can be determined by the agglutination,
precipitin or by indirect FA tests. Unfortunately none of these tests
correlates well with subsequent FA titres of fluorescein isothiocyanate
conjugates made from the serum. They do, however, indicate that an
antibody response has occurred and serve to weed out animals with very
poor titres. I n general, a serum which has a high titre in agglutination
tests or by indirect FA will give a satisfactory conjugate. As a rough guide,
we have found that antiserum with an indirect FA titre of 1 : 500 or greater,
usually yields a usable conjugate. It should be pointed out that there is no
direct correlation between the indirect titre of the whole serum and the
titre of the conjugate.