VI. IDENTIFICATION OF ACTINOMYCES AND ARACHNIA
307
(6) Resuspend cells in 0.1 M PBS, pH 7.6 containing 0.1% NaN3
using 10.0 ml PBS for 1.0 ml cells. Make a slurry of trimethylaminoethyl
(TEAE) cellulose in PBS-NaN3 using 1 .O g TEAE for each 1 a 0 ml packed
cells (measured in (3)). Mix cell suspension with TEAE slurry.
(7) With all reagents at room temperature, pour the TEAE-cellulose
bacterial cell slurry into a column with a 4-1 in plug of nonionic cellulose
powder (Whatman CF-1) at the bottom. When all the slurry is in the
column, allow PBS-NaN3 to flow until effluent and buffer head are free of
cells. Then add another plug of cellulose powder to the top of the column,
followed by a disc of Whatman No. 1 filter paper. Column may be stored
at 22°C
Use of bacterial cell column
(1) For use, wash prepared column with desorbing buffer (0.05 M
NaH2P04 in 1.0% NaCl adjusted to pH 2-3 with HCl), and then equilibrate
with 0.1 M PBS, pH 7.6, followed by 0.05 M PBS, pH 7.4 until a stable
baseline (280 nm on ultraviolet monitor) is obtained.
(2) Add antiserum to the column and elute with pH 7.4 PBS. The
effluent should be monitored by absorbance at 280 nm. After the first
protein fraction (Fl) is eluted, the antibody bound to the column is eluted
with pH 2.3 desorbing buffer (F2). The F1 and F2 fractions are obtained
by pooling the appropriate tubes. Either or both of the fractions may be
conjugated with FITC by standard techniques.
(3) To prepare column for re-use, wash with desorbing buffer and
equilibrate with pH 7.4 PBS. If it is not to be used for several days, wash
with PBS-NaN3 and store at 22°C. For re-use repeat step (1).
Whole unfractionated antiserum, (NH&S04 fractions of antiserum and
IgG fractions of immune serum may be used on bacterial cell columns.
Depending on the absorbing organism and the antibody used, the columns
may be used to prepare pure antibody fractions for conjugation or to
remove cross-reacting antibody from immune serum.
3. Species specijic conjugates
Species specific conjugates for identification consist of pooled sera, each
of which has been absorbed to remove cross-reactions. The individual
components of each pool are absorbed separately and then pooled. The
sera which we have used in the pools are shown in Table IX. T o conserve
absorbed sera, pure culture isolates are usually screened with unabsorbed,
diluted conjugate pools and then checked with absorbed sera as necessary.
The unabsorbed conjugate pools contain the sera listed in Table IX,
diluted so that each serum is present in the pool at its working titre.
Sera prepared as described in Table IX have been used successfully in
307
(6) Resuspend cells in 0.1 M PBS, pH 7.6 containing 0.1% NaN3
using 10.0 ml PBS for 1.0 ml cells. Make a slurry of trimethylaminoethyl
(TEAE) cellulose in PBS-NaN3 using 1 .O g TEAE for each 1 a 0 ml packed
cells (measured in (3)). Mix cell suspension with TEAE slurry.
(7) With all reagents at room temperature, pour the TEAE-cellulose
bacterial cell slurry into a column with a 4-1 in plug of nonionic cellulose
powder (Whatman CF-1) at the bottom. When all the slurry is in the
column, allow PBS-NaN3 to flow until effluent and buffer head are free of
cells. Then add another plug of cellulose powder to the top of the column,
followed by a disc of Whatman No. 1 filter paper. Column may be stored
at 22°C
Use of bacterial cell column
(1) For use, wash prepared column with desorbing buffer (0.05 M
NaH2P04 in 1.0% NaCl adjusted to pH 2-3 with HCl), and then equilibrate
with 0.1 M PBS, pH 7.6, followed by 0.05 M PBS, pH 7.4 until a stable
baseline (280 nm on ultraviolet monitor) is obtained.
(2) Add antiserum to the column and elute with pH 7.4 PBS. The
effluent should be monitored by absorbance at 280 nm. After the first
protein fraction (Fl) is eluted, the antibody bound to the column is eluted
with pH 2.3 desorbing buffer (F2). The F1 and F2 fractions are obtained
by pooling the appropriate tubes. Either or both of the fractions may be
conjugated with FITC by standard techniques.
(3) To prepare column for re-use, wash with desorbing buffer and
equilibrate with pH 7.4 PBS. If it is not to be used for several days, wash
with PBS-NaN3 and store at 22°C. For re-use repeat step (1).
Whole unfractionated antiserum, (NH&S04 fractions of antiserum and
IgG fractions of immune serum may be used on bacterial cell columns.
Depending on the absorbing organism and the antibody used, the columns
may be used to prepare pure antibody fractions for conjugation or to
remove cross-reacting antibody from immune serum.
3. Species specijic conjugates
Species specific conjugates for identification consist of pooled sera, each
of which has been absorbed to remove cross-reactions. The individual
components of each pool are absorbed separately and then pooled. The
sera which we have used in the pools are shown in Table IX. T o conserve
absorbed sera, pure culture isolates are usually screened with unabsorbed,
diluted conjugate pools and then checked with absorbed sera as necessary.
The unabsorbed conjugate pools contain the sera listed in Table IX,
diluted so that each serum is present in the pool at its working titre.
Sera prepared as described in Table IX have been used successfully in