The preparation of an ovine monoclonal to
progesterone
antibody
D. J. Groves, M. J. Sauer, P. Rayment, J. A. Foulkes and B. A. Morris Heterohybridoma Antibody Group, Department of Biochemistry, University of Surrey, Guildford, Surrey gu2 sxh
*MAFF, Cattle Breeding Centre, Church Lane, Shinfield, Reading, Berkshire rg2 9bz (D. J. Groves is now at Cymbus Bioscience Ltd, Epsilon House, Chilworth Research Centre, Southampton,
Hampshire soi 7Ns) (M. J. Sauer is now at Central Veterinary Laboratory, New Haw, Surrey kti5 3nb) (J. A. Foulkes is now at Ridgeway Science Ltd, Rodmore Mill Farm, Alvington, Gloucestershire gli5 6Ah) received 5 January 1990 ABSTRACT
Peripheral blood mononuclear cells were collected from a sheep immunized against progesterone-11\g=a\\x=req-\ hemisuccinate-ovalbumin. Following fusion with NS1 mouse
myeloma
or
heteromyeloma cells,
a
large
number of hybrid colonies was established. These were screened for the production of sheep antibodies to progesterone. Twenty-four cell lines were cloned and one was stabilized. This cell line, O/MP.1A9.D7B2, produced a high-affinity ovine immunoglobulin G1 (dissociation constant 4\m=.\8pmol/l) with a high degree of
INTRODUCTION
Progesterone levels in the milk and blood of cyclic reflect the activity of the corpus luteum and thus provide a precise indicator of ovarian function (Peters & Ball, 1987). Assay of milk progesterone provides an early indication of pregnancy (Booth & Holdsworth, 1976) and can also be used as an aid to oestrus detec¬ tion (Foulkes, Cookson & Sauer, 1982). Both these parameters are important in the efficient management of reproductive performance in the cow as aids to reduce the calving interval. Several assay systems have been described (e.g. Heap, Gwyn, Laing & Walters, 1973; Sauer, Foulkes & Cookson, 1981 ; Van de Weil & Koops, 1986) and more recently rapid and simple enzyme-linked immunosorbant assays (ELISAs) which enable on-site testing have become commercially available. Sheep and goats have provided a source of high-avidity polyclonal anticows
for these assays. A number of mouse monoclonal antibodies (MAbs) to progesterone have also been described. Recent developments in the field of intersera
specificity for progesterone. The antibody was substituted into a competitive enzyme-linked immuno-
sorbant assay for the measurement of progesterone in bovine milk, originally established using an ovine polyclonal antibody, and the results were compared. The monoclonal antibody produced an assay with a lower limit of detection and a greater degree of discrimination than the polyclonal antiserum. Journal ofEndocrinology (1990) 126, 217\p=n-\222
specific fusion have allowed the combination of the desirable characteristics of antisera from large animals with the advantages of MAbs. Monoclonal antibodies offer several advantages over polyclonal antisera as assay reagents. The most important of these are the defined quality of the anti¬ body and its indefinite availability. Ovine antisera against progesterone-1 la-hemisuccinate-ovalbumin (P-llaH-O) were developed as part of investigations into the measurement of progesterone levels in cows' milk using enzyme immunoassay (EIA) (Sauer, 1986). It was intended to immortalize, and perhaps improve, the characteristics of these antibodies by using the animals producing the antisera as sources of sensitized lympho¬ cytes for the production of ovine anti-progesterone MAbs. Although there have been several reports of ovine MAb-secreting cell lines established using inter¬ specific fusion techniques (Groves & Tucker, 1989), most of these lines have subsequently proved to be unstable in long-term culture. The production of ovine MAbs appears to present greater problems than those encountered in the generation of bovine MAbs.
Monoclonal antibodies to progesterone have been
produced using mice (Fanti, Wang & Whitehead, 1981 ; White, Anderson & Daly, 1982; Booman, Tieman, Van der Weil et al. 1984; Fanti & Wang, 1984) and these have been intensively investigated with respect to their
modified Eagle's medium (DMEM) supplemented with 10% (w/v) Myoclone fetal calf serum (PCS) and hypoxanthine, aminopterin and thymidine. Two fusion partners were employed: murine myeloma NSl/l-Ag4-l, and an ovine murine hetero-
to interfere with implantation during preg¬ in the mouse (Wright, Feinstein, Heap et al. nancy 1982; Ellis, Heap, Butchart et al. 1988), their ability to induce anti-idiotypic antibodies (Taussig, Brown, Ellis et al. 1986), and also with respect to antigenantibody complexes (Stura, Arevalo, Feinstein et al. 1987) and their amino acid and nucleotide sequences (Deverson, Berek, Taussig & Feinstein, 1987). This paper reports the comparison of monoclonal murine, polyclonal ovine, and monoclonal ovine antibodies against progesterone with respect to parameters relevant to their potential performance in assay
myeloma (O/MF) (Groves, Clayton & Morris, 1987). The fusion protocol was as described by Groves, Clayton & Morris (1988). The total number of lym¬ phocytes per fusion was 1 IO7 and the ratio of lym¬ phocytes to fusion partner was 5:1. Cells were cloned by limiting dilution into 96-well TC plates over feeder layers of mouse thymocytes.
systems.
(i) Ovine IgG Ovine IgG levels were determined by a sandwich ELISA using a modification of the method of Clayton, Rhind, Groves & Morris (1990). Bovine anti-ovine IgG was immobilized on the assay plates, standards were dilutions of ovine anti-progesterone antiserum in DMEM plus 10% FCS and supernatants were assayed undiluted. The label was horseradish peroxidasebovine anti-ovine IgG. Elimination of the crossreaction with bovine IgG overcame the high back¬ grounds inherent in screens for ovine IgG in FCS-containing medium.
ability
MATERIALS AND METHODS
Reagents All cell culture media and plastics (Nunc) were obtained from Gibco BRL, Paisley, Strathclyde, U.K. Horseradish peroxidase-labelled donkey anti-sheep immunoglobulin (Ig), and unlabelled donkey antisheep Ig, were obtained from Guildhay Antisera Ltd, Guildford, Surrey, U.K. The radiolabel used in the radioimmunoassay (RIA) was [1,2,6,7 tritium] pro¬
gesterone, specific activity 91-8Ci/mmol (Amersham International
reagents
pic, Amersham, Bucks, U.K.).
Other obtained from Sigma Chemical Co.
were
Ltd, Poole, Dorset, U.K. Monoclonal
antibody against progesterone (DB3) was a gift from Professor R. B. Heap (AFRC, Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, Cambs, U.K.). Polyclonal sheep antiserum against progesterone and sensitized lymphocytes for fusion were collected from a Suffolk mouse
wether immunized and boosted with P-l
Immunization
- .
protocol
Primary immunization
was achieved using 2 mg -0 dissolved in 1 ml sterile saline (0-9%, w/v, NaCl) emulsified with 3 ml Freund's complete adju¬ vant and injected into 2 0-5 ml deep intramuscular sites per limb. Boost inoculations consisted of 1 mg conjugate constituted in Freund's incomplete
P-l
adjuvant.
Cell fusion Blood was collected 4 days after boosting and mononuclear cells were isolated on density gradient separ¬ ation medium. Cells were resuspended in Dulbecco's
Screening assays Three EIA systems were employed to detect ovine antibodies in the cell supernatants.
(ii) Ovine anti-progesterone antibodies Polystyrene ELISA plates (Alpha Laboratories) were coated with affinity-purified donkey anti-sheep IgG. Samples or standards (ovine polyclonal progesterone
antiserum diluted in cell culture medium) were incu¬ bated for 2 h at 37 °C. Progesterone-1 la-glucuronidealkaline phosphatase conjugate (Sauer, Foulkes, Worsfold & Morris, 1986) was then added for 2 h at 37 °C. Phosphatase substrate p-nitrophenyl phos¬ phate (pNp; Sigma) was added for 1 h at 37 °C and optical densities (ODs) were read at 405 nm.
(in) Ovine anti-progesterone antibodies ELISA plates were coated with a progesterone 11ahemisuceinate-chicken gammaglobulin conjugate. Samples or standards (as above) were then added. After incubation, horseradish peroxidase-labelled affinity-purified donkey anti-sheep IgG was added for 2 h at 37 °C. Orthophenylenediamine (OPD; Sigma) peroxidase substrate was then added for 30 min at 37 CC in the dark. The enzyme reaction was stopped by the addition of H2S04 and the ODs read at 492 nm. Determination of isotype
Supernatant samples and control media were preincubated with dilutions of murine MAbs to ovine
IgA, IgGl, IgG2, IgM and light chain (obtained from
required to reduce the OD with no unlabelled steroid present by 50%.
transferred to microtitre wells coated with purified ovine IgA, IgG (Gl and G2) or IgM. Following incu¬ bation, wells were incubated with ovine anti-mouse Ig-horseradish peroxidase and the enzyme visualized with OPD/H202 substrate.
Assay of progesterone Rigid polystyrene (NUNC Immuno I) ELISA plates were coated with 200 µ /well of affinity-purified poly¬ clonal donkey antibody against sheep IgG, diluted in carbonate/bicarbonate buffer, at room temperature for 3 h. Plates were washed with 0· 1 mol phosphate buffer/1 (pH 7 ) containing 0T% (w/v) sodium azide and 0-1% (w/v) gelatin (PAS-Gel) and dilutions of polyclonal sheep antibody against progesterone (SI509/16, 1/1000) or monoclonal sheep antibody against progesterone cell culture supernatant (0/MP.1A9.D7B2, 1/1000) in PAS-Gel were added (200 µ /well). Plates were incubated at room tempera¬
Dr . Beh, CSIRO, Division of Animal Health, McMaster Laboratory, NSW, Australia) and then
Determination of affinity The dissociation constants (Kd) of the different anti¬ bodies, and their concentrations, were calculated by a modification of the method described by Groves, Morris, Tan et al. (1987). Antibodies were diluted in phosphate buffer at the following dilutions: ovine M Ab, 1/600, murine M Ab, 1/3000 and ovine poly¬ clonal, 1/9000. Diluted antibodies were incubated with tritiated progesterone (0081 pmol/tube) and increasing amounts of unlabelled progesterone (00039-10 pmol/
tube).
Determination of specificity The specificity of the MAbs and antisera mined in both liquid-phase RIA and ELISA systems.
were
deter¬
solid-phase
Radioimmunoassay for the determination ofspecificity Tritiated progesterone (009 pmol) was incubated over¬ night with a dilution of antibody which bound 50% of the available label and increasing amounts ofunlabelled competing steroid (10"6-103 pmol) diluted in 0-04 mol phosphate buffer/1 (pH 7-4). Bound and free steroids were separated using ice-cold dextran-coated charcoal suspension in phosphate buffer (2-5% (w/v) activated charcoal, 0-25% dextran T70). Antibody-bound radio¬ activity was determined and percentage cross-reactivity was determined as XR P50/C50 100 where P50 and C50 are the masses of progesterone and competitive steroid respectively required to depress the binding with no unlabelled steroid present by 50%. =
ELISA for the determination ofspecificity Specificity in the solid phase was determined by a competitive ELISA. Plates were coated as for screen¬ ing assay (ii) and 200 pi supernatant/well, diluted in assay buffer to give maximal OD in the absence of competing steroids, added for 2 h at 37 °C. Plates were
duplicate dilutions (100 pi) of potentially cross-reacting steroids (10"5-104pmol/well) were washed and
added to wells. Progesterone-alkaline phosphatase label (100 pi) was added to every well and the plates were incubated for 2 h at 37 °C. Plates were washed and incubated with 200 pi pNp substrate at 37 °C for 1 h before reading ODs at 405 nm. Cross-reactivity was calculated with P50 and C50 as the masses of progesterone and competitive steroid respectively
for 2 h. Plates were washed three times with PASGel and 350 µ PAS-Gel/well were added. Plates were sealed and stored at 4 °C until use. Before assay, plates were inverted and tapped dry. Ten microlitres milk samples or standards were added to wells followed by 200 µ progesterone-11 glucuronide-alkaline phosphatase conjugate diluted in PAS-Gel. Plates were incubated for 3 h at room temperature before washing with PAS-Gel and the addition of 200 µ pNp substrate/well. Plates were incubated for 1 h at 40 °C in a water bath and ODs were read at 405 nm. ture
RESULTS
Fusion
Wells were screened for visible colonies 7 and 24 days after fusion. Cell supernatants were screened for the presence of ovine IgG and ovine progesterone anti¬
bodies using assays (i), (ii) and (iii) 10 and 24 days after fusion. Fusions conducted using O/MF heteromyelomas resulted in higher fusion efficiencies than those using NS1 cells. A total of 24 wells were cloned between 26 and 39 days after fusion. At 57 days after fusion one NS1-derived line, 0/MP.1A9, remained positive in screening with 14-6% of cloned wells positive for pro¬
gesterone antibodies (assay ii). Four of these lines
subcloned and one line, 0/MP.1A9.D7, was selected on the grounds of growth and secretion rates for further cloning, expansion and characterization. The doubling-time of the line suspended in DMEM plus 10% FCS was approximately 26 h. In static batch culture the peak cell density achieved was 1-6 IO6 cells/ml and a peak antibody concentration of 1020 µg/ml was obtained after 7 days. Production rates for static batch cultures over 5 days were 5-10 µg/106 cells per 24 h. Karyotyping revealed the chromosome were
1. Percentage cross-reactivities of different steroids with ovine monoclonal anti-progesterone antibody 0/MP.1A9.D7B2 (O/MP), ovine anti-progesterone antiserum S1509/16 (ShaP) and mouse monoclonal anti-progesterone antibody (DB3) in solid (ELISA)- and liquid (RIA)-phase assay table
systems Cross reactivities (%) DB3
ShaP
O/MP ELISA
RIA
ELISA
RIA
ELISA
RIA
100 48-8 23 1 56-6 5-3 4-9
100 69-3 530 60-5 21 2-7
5-4 0-5 0-3
80
progesterone
antibody
D. J. Groves, M. J. Sauer, P. Rayment, J. A. Foulkes and B. A. Morris Heterohybridoma Antibody Group, Department of Biochemistry, University of Surrey, Guildford, Surrey gu2 sxh
*MAFF, Cattle Breeding Centre, Church Lane, Shinfield, Reading, Berkshire rg2 9bz (D. J. Groves is now at Cymbus Bioscience Ltd, Epsilon House, Chilworth Research Centre, Southampton,
Hampshire soi 7Ns) (M. J. Sauer is now at Central Veterinary Laboratory, New Haw, Surrey kti5 3nb) (J. A. Foulkes is now at Ridgeway Science Ltd, Rodmore Mill Farm, Alvington, Gloucestershire gli5 6Ah) received 5 January 1990 ABSTRACT
Peripheral blood mononuclear cells were collected from a sheep immunized against progesterone-11\g=a\\x=req-\ hemisuccinate-ovalbumin. Following fusion with NS1 mouse
myeloma
or
heteromyeloma cells,
a
large
number of hybrid colonies was established. These were screened for the production of sheep antibodies to progesterone. Twenty-four cell lines were cloned and one was stabilized. This cell line, O/MP.1A9.D7B2, produced a high-affinity ovine immunoglobulin G1 (dissociation constant 4\m=.\8pmol/l) with a high degree of
INTRODUCTION
Progesterone levels in the milk and blood of cyclic reflect the activity of the corpus luteum and thus provide a precise indicator of ovarian function (Peters & Ball, 1987). Assay of milk progesterone provides an early indication of pregnancy (Booth & Holdsworth, 1976) and can also be used as an aid to oestrus detec¬ tion (Foulkes, Cookson & Sauer, 1982). Both these parameters are important in the efficient management of reproductive performance in the cow as aids to reduce the calving interval. Several assay systems have been described (e.g. Heap, Gwyn, Laing & Walters, 1973; Sauer, Foulkes & Cookson, 1981 ; Van de Weil & Koops, 1986) and more recently rapid and simple enzyme-linked immunosorbant assays (ELISAs) which enable on-site testing have become commercially available. Sheep and goats have provided a source of high-avidity polyclonal anticows
for these assays. A number of mouse monoclonal antibodies (MAbs) to progesterone have also been described. Recent developments in the field of intersera
specificity for progesterone. The antibody was substituted into a competitive enzyme-linked immuno-
sorbant assay for the measurement of progesterone in bovine milk, originally established using an ovine polyclonal antibody, and the results were compared. The monoclonal antibody produced an assay with a lower limit of detection and a greater degree of discrimination than the polyclonal antiserum. Journal ofEndocrinology (1990) 126, 217\p=n-\222
specific fusion have allowed the combination of the desirable characteristics of antisera from large animals with the advantages of MAbs. Monoclonal antibodies offer several advantages over polyclonal antisera as assay reagents. The most important of these are the defined quality of the anti¬ body and its indefinite availability. Ovine antisera against progesterone-1 la-hemisuccinate-ovalbumin (P-llaH-O) were developed as part of investigations into the measurement of progesterone levels in cows' milk using enzyme immunoassay (EIA) (Sauer, 1986). It was intended to immortalize, and perhaps improve, the characteristics of these antibodies by using the animals producing the antisera as sources of sensitized lympho¬ cytes for the production of ovine anti-progesterone MAbs. Although there have been several reports of ovine MAb-secreting cell lines established using inter¬ specific fusion techniques (Groves & Tucker, 1989), most of these lines have subsequently proved to be unstable in long-term culture. The production of ovine MAbs appears to present greater problems than those encountered in the generation of bovine MAbs.
Monoclonal antibodies to progesterone have been
produced using mice (Fanti, Wang & Whitehead, 1981 ; White, Anderson & Daly, 1982; Booman, Tieman, Van der Weil et al. 1984; Fanti & Wang, 1984) and these have been intensively investigated with respect to their
modified Eagle's medium (DMEM) supplemented with 10% (w/v) Myoclone fetal calf serum (PCS) and hypoxanthine, aminopterin and thymidine. Two fusion partners were employed: murine myeloma NSl/l-Ag4-l, and an ovine murine hetero-
to interfere with implantation during preg¬ in the mouse (Wright, Feinstein, Heap et al. nancy 1982; Ellis, Heap, Butchart et al. 1988), their ability to induce anti-idiotypic antibodies (Taussig, Brown, Ellis et al. 1986), and also with respect to antigenantibody complexes (Stura, Arevalo, Feinstein et al. 1987) and their amino acid and nucleotide sequences (Deverson, Berek, Taussig & Feinstein, 1987). This paper reports the comparison of monoclonal murine, polyclonal ovine, and monoclonal ovine antibodies against progesterone with respect to parameters relevant to their potential performance in assay
myeloma (O/MF) (Groves, Clayton & Morris, 1987). The fusion protocol was as described by Groves, Clayton & Morris (1988). The total number of lym¬ phocytes per fusion was 1 IO7 and the ratio of lym¬ phocytes to fusion partner was 5:1. Cells were cloned by limiting dilution into 96-well TC plates over feeder layers of mouse thymocytes.
systems.
(i) Ovine IgG Ovine IgG levels were determined by a sandwich ELISA using a modification of the method of Clayton, Rhind, Groves & Morris (1990). Bovine anti-ovine IgG was immobilized on the assay plates, standards were dilutions of ovine anti-progesterone antiserum in DMEM plus 10% FCS and supernatants were assayed undiluted. The label was horseradish peroxidasebovine anti-ovine IgG. Elimination of the crossreaction with bovine IgG overcame the high back¬ grounds inherent in screens for ovine IgG in FCS-containing medium.
ability
MATERIALS AND METHODS
Reagents All cell culture media and plastics (Nunc) were obtained from Gibco BRL, Paisley, Strathclyde, U.K. Horseradish peroxidase-labelled donkey anti-sheep immunoglobulin (Ig), and unlabelled donkey antisheep Ig, were obtained from Guildhay Antisera Ltd, Guildford, Surrey, U.K. The radiolabel used in the radioimmunoassay (RIA) was [1,2,6,7 tritium] pro¬
gesterone, specific activity 91-8Ci/mmol (Amersham International
reagents
pic, Amersham, Bucks, U.K.).
Other obtained from Sigma Chemical Co.
were
Ltd, Poole, Dorset, U.K. Monoclonal
antibody against progesterone (DB3) was a gift from Professor R. B. Heap (AFRC, Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, Cambs, U.K.). Polyclonal sheep antiserum against progesterone and sensitized lymphocytes for fusion were collected from a Suffolk mouse
wether immunized and boosted with P-l
Immunization
- .
protocol
Primary immunization
was achieved using 2 mg -0 dissolved in 1 ml sterile saline (0-9%, w/v, NaCl) emulsified with 3 ml Freund's complete adju¬ vant and injected into 2 0-5 ml deep intramuscular sites per limb. Boost inoculations consisted of 1 mg conjugate constituted in Freund's incomplete
P-l
adjuvant.
Cell fusion Blood was collected 4 days after boosting and mononuclear cells were isolated on density gradient separ¬ ation medium. Cells were resuspended in Dulbecco's
Screening assays Three EIA systems were employed to detect ovine antibodies in the cell supernatants.
(ii) Ovine anti-progesterone antibodies Polystyrene ELISA plates (Alpha Laboratories) were coated with affinity-purified donkey anti-sheep IgG. Samples or standards (ovine polyclonal progesterone
antiserum diluted in cell culture medium) were incu¬ bated for 2 h at 37 °C. Progesterone-1 la-glucuronidealkaline phosphatase conjugate (Sauer, Foulkes, Worsfold & Morris, 1986) was then added for 2 h at 37 °C. Phosphatase substrate p-nitrophenyl phos¬ phate (pNp; Sigma) was added for 1 h at 37 °C and optical densities (ODs) were read at 405 nm.
(in) Ovine anti-progesterone antibodies ELISA plates were coated with a progesterone 11ahemisuceinate-chicken gammaglobulin conjugate. Samples or standards (as above) were then added. After incubation, horseradish peroxidase-labelled affinity-purified donkey anti-sheep IgG was added for 2 h at 37 °C. Orthophenylenediamine (OPD; Sigma) peroxidase substrate was then added for 30 min at 37 CC in the dark. The enzyme reaction was stopped by the addition of H2S04 and the ODs read at 492 nm. Determination of isotype
Supernatant samples and control media were preincubated with dilutions of murine MAbs to ovine
IgA, IgGl, IgG2, IgM and light chain (obtained from
required to reduce the OD with no unlabelled steroid present by 50%.
transferred to microtitre wells coated with purified ovine IgA, IgG (Gl and G2) or IgM. Following incu¬ bation, wells were incubated with ovine anti-mouse Ig-horseradish peroxidase and the enzyme visualized with OPD/H202 substrate.
Assay of progesterone Rigid polystyrene (NUNC Immuno I) ELISA plates were coated with 200 µ /well of affinity-purified poly¬ clonal donkey antibody against sheep IgG, diluted in carbonate/bicarbonate buffer, at room temperature for 3 h. Plates were washed with 0· 1 mol phosphate buffer/1 (pH 7 ) containing 0T% (w/v) sodium azide and 0-1% (w/v) gelatin (PAS-Gel) and dilutions of polyclonal sheep antibody against progesterone (SI509/16, 1/1000) or monoclonal sheep antibody against progesterone cell culture supernatant (0/MP.1A9.D7B2, 1/1000) in PAS-Gel were added (200 µ /well). Plates were incubated at room tempera¬
Dr . Beh, CSIRO, Division of Animal Health, McMaster Laboratory, NSW, Australia) and then
Determination of affinity The dissociation constants (Kd) of the different anti¬ bodies, and their concentrations, were calculated by a modification of the method described by Groves, Morris, Tan et al. (1987). Antibodies were diluted in phosphate buffer at the following dilutions: ovine M Ab, 1/600, murine M Ab, 1/3000 and ovine poly¬ clonal, 1/9000. Diluted antibodies were incubated with tritiated progesterone (0081 pmol/tube) and increasing amounts of unlabelled progesterone (00039-10 pmol/
tube).
Determination of specificity The specificity of the MAbs and antisera mined in both liquid-phase RIA and ELISA systems.
were
deter¬
solid-phase
Radioimmunoassay for the determination ofspecificity Tritiated progesterone (009 pmol) was incubated over¬ night with a dilution of antibody which bound 50% of the available label and increasing amounts ofunlabelled competing steroid (10"6-103 pmol) diluted in 0-04 mol phosphate buffer/1 (pH 7-4). Bound and free steroids were separated using ice-cold dextran-coated charcoal suspension in phosphate buffer (2-5% (w/v) activated charcoal, 0-25% dextran T70). Antibody-bound radio¬ activity was determined and percentage cross-reactivity was determined as XR P50/C50 100 where P50 and C50 are the masses of progesterone and competitive steroid respectively required to depress the binding with no unlabelled steroid present by 50%. =
ELISA for the determination ofspecificity Specificity in the solid phase was determined by a competitive ELISA. Plates were coated as for screen¬ ing assay (ii) and 200 pi supernatant/well, diluted in assay buffer to give maximal OD in the absence of competing steroids, added for 2 h at 37 °C. Plates were
duplicate dilutions (100 pi) of potentially cross-reacting steroids (10"5-104pmol/well) were washed and
added to wells. Progesterone-alkaline phosphatase label (100 pi) was added to every well and the plates were incubated for 2 h at 37 °C. Plates were washed and incubated with 200 pi pNp substrate at 37 °C for 1 h before reading ODs at 405 nm. Cross-reactivity was calculated with P50 and C50 as the masses of progesterone and competitive steroid respectively
for 2 h. Plates were washed three times with PASGel and 350 µ PAS-Gel/well were added. Plates were sealed and stored at 4 °C until use. Before assay, plates were inverted and tapped dry. Ten microlitres milk samples or standards were added to wells followed by 200 µ progesterone-11 glucuronide-alkaline phosphatase conjugate diluted in PAS-Gel. Plates were incubated for 3 h at room temperature before washing with PAS-Gel and the addition of 200 µ pNp substrate/well. Plates were incubated for 1 h at 40 °C in a water bath and ODs were read at 405 nm. ture
RESULTS
Fusion
Wells were screened for visible colonies 7 and 24 days after fusion. Cell supernatants were screened for the presence of ovine IgG and ovine progesterone anti¬
bodies using assays (i), (ii) and (iii) 10 and 24 days after fusion. Fusions conducted using O/MF heteromyelomas resulted in higher fusion efficiencies than those using NS1 cells. A total of 24 wells were cloned between 26 and 39 days after fusion. At 57 days after fusion one NS1-derived line, 0/MP.1A9, remained positive in screening with 14-6% of cloned wells positive for pro¬
gesterone antibodies (assay ii). Four of these lines
subcloned and one line, 0/MP.1A9.D7, was selected on the grounds of growth and secretion rates for further cloning, expansion and characterization. The doubling-time of the line suspended in DMEM plus 10% FCS was approximately 26 h. In static batch culture the peak cell density achieved was 1-6 IO6 cells/ml and a peak antibody concentration of 1020 µg/ml was obtained after 7 days. Production rates for static batch cultures over 5 days were 5-10 µg/106 cells per 24 h. Karyotyping revealed the chromosome were
1. Percentage cross-reactivities of different steroids with ovine monoclonal anti-progesterone antibody 0/MP.1A9.D7B2 (O/MP), ovine anti-progesterone antiserum S1509/16 (ShaP) and mouse monoclonal anti-progesterone antibody (DB3) in solid (ELISA)- and liquid (RIA)-phase assay table
systems Cross reactivities (%) DB3
ShaP
O/MP ELISA
RIA
ELISA
RIA
ELISA
RIA
100 48-8 23 1 56-6 5-3 4-9
100 69-3 530 60-5 21 2-7
5-4 0-5 0-3
80
