VI. IDENTIFICATION OF ACTINOMYCES AND ARACHNIA
297
(h) Add enough 0.85% NaCl to bring the total volume to twice the
(i) Allow to react at 25°C for 2-5 h without shaking.
(j) Remove any precipitate which forms by centrifugation and dialyse
against pH 7.6 phosphate-buffered saline to remove unreacted fluorescein.
Unreacted fluorescein can also be removed by passage through a Sephadex
column.
(k) Centrifuge again if necessary. Add merthiolate (1 : 10 000) and
store at 4°C or freeze in small aliquots. Repeated freezing and thawing
should be avoided.
3. Evaluation of the conjugate
Performance assays of conjugates are done by staining homologous and
heterologous antigens (in this case cell suspensions of Actinomyces) with
two-fold dilutions of conjugate. Background staining in such smears should
be noted as a guide to the presence of unreacted fluorescein and to possible
non-specific staining. Evaluation of non-specific staining can be done by
staining mammalian cell culture smears (Pittman et al., 1967) or by
staining leukcocytes from venous blood (Holmberg and Forsum, 1973).
FA staining titres which allow dilution of the conjugate are highly desirable
since such dilution is probably the most efficient method of reducing or
removing true non-specific staining. Cross-reactions due to common
antigens in the bacteria can be removed by appropriate absorption of the
conjugate.
Other tests should also be done on conjugates, if possible. The F/P
ratio should be determined using the method of Wells et al. (1966) or that
of McKinney et al. (see Hebert et al., 1972). Cellulose acetate strip electrophoresis (Hebert et al., 1971 ; Hebert et al., 1972) yields valuable information as to the purity of the globulin labelled and the presence of unreacted FITC.
Optimal F/P ratios for Actinomyces conjugates are not well defined. The
optimal ratio for conjugates made by direct labelling of (NH&S04 fractionated antiserum is in the range of 15-20 (Bragg, pers. comm.). Our
experience also suggests that while conjugates with F/P ratios of approximately 10 have good titres, those with ratios between 15 and 20 usually
have higher staining titres. It should be remembered that F/P ratios
determined on conjugates prepared by this method reflect average labelling
of the globulin since the globulins are not homogeneously labelled.
Holmberg and Forsum (1973) determined the optimal F/P ratio for
Actinomyces conjugates. In this case, purified IgG was labelled and then
fractionated on a DEAE cellulose column to yield homogeneous fractions
with varying F,/P ratios. In this study, conjugates with F/P ratios of 10 or
original volume of globulin; mix gently.
297
(h) Add enough 0.85% NaCl to bring the total volume to twice the
(i) Allow to react at 25°C for 2-5 h without shaking.
(j) Remove any precipitate which forms by centrifugation and dialyse
against pH 7.6 phosphate-buffered saline to remove unreacted fluorescein.
Unreacted fluorescein can also be removed by passage through a Sephadex
column.
(k) Centrifuge again if necessary. Add merthiolate (1 : 10 000) and
store at 4°C or freeze in small aliquots. Repeated freezing and thawing
should be avoided.
3. Evaluation of the conjugate
Performance assays of conjugates are done by staining homologous and
heterologous antigens (in this case cell suspensions of Actinomyces) with
two-fold dilutions of conjugate. Background staining in such smears should
be noted as a guide to the presence of unreacted fluorescein and to possible
non-specific staining. Evaluation of non-specific staining can be done by
staining mammalian cell culture smears (Pittman et al., 1967) or by
staining leukcocytes from venous blood (Holmberg and Forsum, 1973).
FA staining titres which allow dilution of the conjugate are highly desirable
since such dilution is probably the most efficient method of reducing or
removing true non-specific staining. Cross-reactions due to common
antigens in the bacteria can be removed by appropriate absorption of the
conjugate.
Other tests should also be done on conjugates, if possible. The F/P
ratio should be determined using the method of Wells et al. (1966) or that
of McKinney et al. (see Hebert et al., 1972). Cellulose acetate strip electrophoresis (Hebert et al., 1971 ; Hebert et al., 1972) yields valuable information as to the purity of the globulin labelled and the presence of unreacted FITC.
Optimal F/P ratios for Actinomyces conjugates are not well defined. The
optimal ratio for conjugates made by direct labelling of (NH&S04 fractionated antiserum is in the range of 15-20 (Bragg, pers. comm.). Our
experience also suggests that while conjugates with F/P ratios of approximately 10 have good titres, those with ratios between 15 and 20 usually
have higher staining titres. It should be remembered that F/P ratios
determined on conjugates prepared by this method reflect average labelling
of the globulin since the globulins are not homogeneously labelled.
Holmberg and Forsum (1973) determined the optimal F/P ratio for
Actinomyces conjugates. In this case, purified IgG was labelled and then
fractionated on a DEAE cellulose column to yield homogeneous fractions
with varying F,/P ratios. In this study, conjugates with F/P ratios of 10 or
original volume of globulin; mix gently.