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M. A. GERENCSER
C. Preparation of absorbed conjugates
1. Batch absorption method
Cells for absorption are grown in TSB broth for 3-5 days, harvested
by centrifugation, washed twice in FTA buffer and resuspended in a very
small amount of buffer. This heavy suspension is stored at 4°C until
used, at which time the cells are packed by centrifugation and measured.
Conjugated antiserum (1.0 ml to 0.1 ml packed cells) is added, the cells
are suspended evenly in the serum and the mixture incubated at 50°C for 1
h. After overnight refrigeration, the conjugate is centrifuged to remove the
cells and the process repeated with fresh cells for a total of three absorptions.
The conjugate is then tested with the absorbing strain and if absorption is
complete the serum is titred with the homologous strain. Absorbed serum
is clarified by high speed centrifugation and preserved with merthiolate
(1 : 10 000). If desired, the serum may be filter sterilised and stored frozen
without a preservative.
2. Column absorption method
McKinney and Thacker (1976a, b) described a bacterial cell column
absorption method for preparing serotype specific Streptococcus mutans
conjugates. Bragg et a1 (1977) have adapted the column absorption technique for the preparation of species and serotype specific reagents for
Actinomyces, Arachnia, and Rothia. The following procedure for the
preparation of immunoabsorbent columns is based on the report of Bragg
et al. (1977) and on additional details kindly supplied by Sandra Bragg.
Preparation of bacterial-cell columns
(1) Add formalin (1% final concentration) to a broth culture of the
organism to be used and allow the culture to stand at least 2 h.
(2) Harvest cells by centrifugation and wash once in 0-S5y0 saline
containing 0.5yo formalin.
(3) Resuspend cells in acetone and centrifuge at 1000 rev/min for
15 min. Measure packed cells and decant acetone.
(4) Resuspend cells in 0.01 M phosphate buffered saline (PBS), pH 7.4,
containing 1 : 10 000 merthiolate, using 10.0 ml PBS for each 1.0 ml packed
cells.
( 5 ) Label cells with F I T C (5 mg FITC/mg packed cells) by mixing a
0.1% solution of F I T C in 0.1 M NaZHP04 with the cell slurry. Raise p H
to 9.5 and incubate at room temperature overnight. Centrifuge, decant
excess F I T C and wash labelled cells once in PBS, pH 7.4.
Labelling of cells with F I T C may be omitted but labelled cells adhere
to columns better than do unlabelled cells.
M. A. GERENCSER
C. Preparation of absorbed conjugates
1. Batch absorption method
Cells for absorption are grown in TSB broth for 3-5 days, harvested
by centrifugation, washed twice in FTA buffer and resuspended in a very
small amount of buffer. This heavy suspension is stored at 4°C until
used, at which time the cells are packed by centrifugation and measured.
Conjugated antiserum (1.0 ml to 0.1 ml packed cells) is added, the cells
are suspended evenly in the serum and the mixture incubated at 50°C for 1
h. After overnight refrigeration, the conjugate is centrifuged to remove the
cells and the process repeated with fresh cells for a total of three absorptions.
The conjugate is then tested with the absorbing strain and if absorption is
complete the serum is titred with the homologous strain. Absorbed serum
is clarified by high speed centrifugation and preserved with merthiolate
(1 : 10 000). If desired, the serum may be filter sterilised and stored frozen
without a preservative.
2. Column absorption method
McKinney and Thacker (1976a, b) described a bacterial cell column
absorption method for preparing serotype specific Streptococcus mutans
conjugates. Bragg et a1 (1977) have adapted the column absorption technique for the preparation of species and serotype specific reagents for
Actinomyces, Arachnia, and Rothia. The following procedure for the
preparation of immunoabsorbent columns is based on the report of Bragg
et al. (1977) and on additional details kindly supplied by Sandra Bragg.
Preparation of bacterial-cell columns
(1) Add formalin (1% final concentration) to a broth culture of the
organism to be used and allow the culture to stand at least 2 h.
(2) Harvest cells by centrifugation and wash once in 0-S5y0 saline
containing 0.5yo formalin.
(3) Resuspend cells in acetone and centrifuge at 1000 rev/min for
15 min. Measure packed cells and decant acetone.
(4) Resuspend cells in 0.01 M phosphate buffered saline (PBS), pH 7.4,
containing 1 : 10 000 merthiolate, using 10.0 ml PBS for each 1.0 ml packed
cells.
( 5 ) Label cells with F I T C (5 mg FITC/mg packed cells) by mixing a
0.1% solution of F I T C in 0.1 M NaZHP04 with the cell slurry. Raise p H
to 9.5 and incubate at room temperature overnight. Centrifuge, decant
excess F I T C and wash labelled cells once in PBS, pH 7.4.
Labelling of cells with F I T C may be omitted but labelled cells adhere
to columns better than do unlabelled cells.