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One-step purification of mouse monoclonal antibodies by mass ion exchange chromatography on Zetaprep
Journal of Immunological Methods, 99 (1987) 229-233 Elsevier
229
JIM 04349
One-step purification of mouse monoclonal antibodies by mass ion exchange chromatography on Zetaprep * F . D . M e n o z z i 1, p. V a n d e r p o o r t e n 2, C. Dejaiffe 2 and A.O.A. Miller 1 l UnitO de Biotechnologie Appliqu~e, FacultO de M~decine, Universit~ de l'Etat, Avenue du Champ de Mars, 24, 7000 Mons, and 2 Biologie et Biotechnologie S.A., Chaussbe de Binche, 101, 7000 Mons, Belgium Received 3 December 1986, revised received 21 January 1987, accepted 26 January 1987).
A rapid one-step purification procedure was developed to isolate mouse monoclonal antibodies present in the growth medium of hybridoma cultures. The procedure, based on a sulfopropyl mass ion exchange chromatography, yields considerable amounts of purified monoclonal antibody. The immunoglobulins are essentially free of transferrin and albumin, as determined by SDS-polyacrylamide gel electrophoresis. Key words: Monoclonal antibody purification; Zetaprep, sulfopropyl
Introduction Compared to monoclonal antibodies obtained from the ascitic fluid of mice injected intraperitoneally with hybridomas, which may be contaminated with various pathogens or contain proteolytic enzymes, those isolated from the growth medium used to cultivate the hybridomas in vitro - especially the synthetic ones - are less complex and therefore easier to purify. The main difficulty of this approach results from the very low concentrations at which these molecules are present and the consequent need to concentrate as well as purify them. In this work, we have purified mouse monoCorrespondence to: A.O.A. Miller, Unit~ de Biotechnologie Appliqure, Facult6 de Mrdecine, Universit~ de l'Etat, Avenue du Champ de Mars, 24, 7000 Mons, Belgium. * Work having partially benefited from the research-development project conducted with Biologie et Biotechnologie S.A. within the framework of an A.R. 123 PME agreement. Abbreviations: PBS, phosphate-buffered saline; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; SDS-Page, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SP, sulfopropyl.
clonal antibodies directly from large volumes of hybridoma culture medium, using the 2223 Zetaprep sulfopropyl mass ion exchange matrix recently developed by A M F Molecular Separations Division for LKB Produkter.
Materials and methods
Hybridomas The two hybridoma cell lines used in this work, Bambou 819 and Mompou 001 result from the polyethylene glycol 1000-mediated fusion of the SP20 murine myeloma cells with spleen cells of C3H mice immunized either with ferritin purified from HeLa cells (Miller, 1968; Kadouche et al., 1982) or with Chlamydia outer membrane complex (COMC) prepared as described by Caldwell (Caldwell et al., 1981). High titers (6-10 /~g/ml) of monoclonal antibodies (anti-MOMP antibodies) were obtained by growing the hybridomas at high cell densities in 200 cm z glass Roux flasks in supplemented D M E M containing 1% heat-inactivated newborn calf serum and 19% IBF Ultroser HY. After 2 days, the Roux
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
230 flasks were violently shaken. The medium and the cells they contain were transferred into empty Roux flasks from which the monoclonal antibody was removed following a further 24 h incubation at 37 o C. The hybridomas remaining in the initial Roux flasks were refed with fresh medium for re-use 2 days after. Sulfopropyl Zetaprep ion exchange purification of monoclonal antibodies In both of the two elution protocols which have been routinely used, the AMF Zetaprep 250 SP cartridge cast in its housing was first equilibrated with two bed volumes (500 ml) of 200 mM acetate buffer pH 5.5 at a flow rate of 300 m l / h . Then, between 300 and 450 ml of the culture supernatant from Mompou 001 hybridomas previously adjusted to pH 5.5 with HC1 were applied at a reduced flow rate of 150 m l / h (the same flow rate was maintained during all subsequent steps in both protocols) and 6 ml samples were collected. In the stepwise elution protocol, a first wash of the cartridge with 300 ml 200 mM acetate buffer p H 5.5 was followed by 250 ml of 200 mM TrisHCI pH 8.00 buffer containing successively 0, 0.2, 0.4 and 1 M NaC1. The second strategy adopted a linear 760 ml NaC1 gradient (from 0 to 0.5 M) buffered by 100 mM Tris-HC1 pH 8.00, using the LKB 11300 Ultrograd gradient mixer. All manipulations took place at room temperature. The immunoglobulin (Ig), containing fractions detected by ELISA, were pooled and an equal volume of saturated ammonium sulfate solution added in order to reach half saturation levels. After one night at 4 ° C , the precipitated IgG were collected following a 1 h centrifugation at 20000 x gay in a number 14 rotor of a preparative J-21B Beckman centrifuge run at 4°C. The Ig pellet was redissolved in 2 ml PBS, the resulting solution dialyzed for 14 h against 200 vols. of 20 mM Tris-HC1 buffer pH 7.2 and the dissolved Ig kept frozen at - 90 ° C. E L I S A detection of monoclonal antibody-containing fractions Using the method developed by Goding (Goding, 1980) the wells of 96-well PVC microtiter plates were filled with 50 #1 of a 20 # g / m l solution of COMC dissolved in PBS. Following the
usual washes and saturation of fixation sites with bovine serum albumin, the wells were filled with 50 ffl of the test solution and incubated. After supplementary washes, immobilized anti-MOMP antibodies were reacted with a 1/250 diluted alkaline phosphatase-conjugated goat anti-mouse IgG solution and their presence revealed by the addition of paranitrophenol phosphate (reading the optical density in each well at 405 nm with a Flow Titertek Multiskan). S D S gel electrophoresis - Column capacity determination - Protein determinations Analysis of the purity of the proteins eluted from the SP Zetaprep cartridge was performed by SDS-polyacrylamide gel electrophoresis on linear 4-22% gradient gels (Laemmli, 1970) using Coomassie brillant R250 to stain the separated bands. Maximum loading capacity of the column was determined by continuously charging the support with the culture supernatant until antibody molecules were detected by ELISA in the flow-through. Protein determinations were performed as described by Lowry (Lowry et al., 1951).
Results
Stepwise elution A Zetaprep ion exchange cartridge was loaded with 300 ml of Mompou 001 culture supernatant at p H 5.5 and washed with pH 5.5 buffer. It was then washed with a succession of increased concentrations of NaC1 in 200 mM Tris pH 8.00 as described in Fig. 1. The Ig (IgG1) was eluted in the first wash (zero NaC1). Polyacrylamide gel electrophoresis performed under denaturing conditions in the presence of SDS (SDS-PAGE) revealed the Ig to be contaminated by a material whose electrophoretic mobility was similar to that of albumin (Fig. 2). No contamination with transferrin was detected. Gradient elution In order to improve the purification of Ig, elution from the column was started at a lower ionic strength (100 mM Tris pH 8.00) with a continuous gradient of increasing NaC1 concentra-
231
6~.
E
E=.
Ex
E=
/2
l: I t
' "
°o
'°!
i
.... 5 25
50
75
I00
125
Fig. 1. Stepwise elution profile of 300 ml Mompou 001 supernatant on SP Zetaprep 250. The protein elution was monitored by UV recording at 280 nm (O O) and the antibody elution was determined by a specific ELISA (O O).
150
FRACTION NUMBER (6ml)
1
2
3
4
tion. With this technique, the anti-MOMP Ig was eluted at 250 mM NaC1 (Fig. 3). SDS-PAGE analysis (Fig. 2) of the Ig eluted in this way showed only two bands corresponding to the heavy and fight chains. Neither albumin nor transferrin could be detected. Table I summarizes the salient characteristics of this mode of elution.
5
6
7
W
N
17 Fig. 2. SDS-PAGE analysis of the fractions eluted from the SP-Zetaprep. Lane I: Mompou 001 culture supematant. Lane 2: Bovine serum albumin. Lane 3: Mompou 001 monoclonal antibodies obtained by stepwise elution from three independent experiments (as in lane 5), pooled and submitted to a new stepwise elution chromatography on SP Zetaprep. Little albumin contamination is visible. Lane 4: Fractions 129 to 142 from the gradient elution of 300 ml Bambou 819 culture supernatant. Lane 5: Fractions 90-96 from the stepwise elution of 300 ml Mompou 001 culture supernatant. Lane 6: Fractions 130-139 from the gradient elution of 300 ml Mompou 001 culture supernatant. Lane 7: Molecular weight markers: phosphorylase B (94 KD), albumin (67 KD), ovalbumin (43 KD), carbonic anhydrase (30 KD), heavy and light ferritin sub-units (19 and 17 KD).
232 TABLE I C O M P A R A T I V E S U M M A R Y O F T H E T W O M O N O C L O N A L A N T I B O D Y P U R I F I C A T I O N S ON SP Z E T A P R E P 250 F R O M IN VITRO C U L T U R E S U P E R N A T A N T S OF T H E B A M B O U 819 A N D M O M P O U 001 H Y B R I D O M A S ( G R A D I E N T SALT ELUTION) Cell line
Monoclonal IgG secreted (/~g/ml)
Hybridoma supernatant loaded on the SP Zetaprep (ml)
Monoclonal antibody recovery (%)
Purification ~ factor
Concentration b factor
Bambou 819 clone M o m p o u 001 clone
8 6
300 300
89 90
15 10
3 3
Purification factor: calculated from the relation A z / A o × B o / B z where A z and A o are the mean concentration ( ~ g / m l ) of M o m p o u IgG in the eluate from the Zetaprep ( A z ) and in the culture supernatant (Ao), B z and Bo being the mean protein concentration (t*g/ml) in the eluate from the Zetaprep ( B z ) and in the culture supernatant (Bo). b Concentration factor: calculated as the ratio of the volume of culture supernatant put on the Zetaprep cartridge divided by the volume of the eluate containing the IgG. a
25
50
75
IN
12S
150
175
FRACTION NUMBER (6ml)
Fig. 3. Gradient elution profile of 300 ml M o m p o u 001 supernatant on SP Zetaprep 250. A linear gradient salt from 0 to 500 m M NaC1 was used to remove the proteins adsorbed to the matrix ( I m). The protein (O O) and monoclonal antibody (O O) elutions were monitored as in Fig. 1. The flow rate was maintained constant at 150 m l / h and the cartridge developed as described in the materials and methods section.
Tris pH 8.0, 1 M NaC1 and 4 bed volumes of 200 m M acetate buffer pH 5.5) takes approximately the same time, as many as two successive cycles of purification and regeneration can be performed per 24 h allowing as much as 3 mg of pure monoclonal antibody to be obtained daily. Extension of this technology to the purification of IgG1 molecules, secreted by the murine Bambou clone 819 against ferritin extracted from HeLa ceils, gave identical results (except for the elution of the antibody at 260 mM NaC1). Gradient elution is not as well suited as stepwise elution to operations on an industrial scale. Our results show that stepwise elution yields Ig contaminated by albumin (present in the newborn calf serum added to the growth medium). It is possible that careful optimization of the latter procedure could eliminate the observed contamination. Alternatively, the high flow rate of the Zetaprep cartridge could be used for the large scale removal of albumin by a stepwise elution protocol on a SP Zetaprep 250 followed downstream by a Protein A-Zetaprep cartridge.
Discussion
Acknowledgements
SP Zetaprep ion exchange chromatography allows easy, rapid and almost quantitative recovery of murine monoclonal antibodies produced by hybridomas grown in dilute serum medium. As much as 500 ml of Ig-containing growth medium can be passed through the cartridge in less than 5 h. Because regeneration (1 bed volume of 100 mM
We are much indebted to Mr. J. Jansseune (LKB, Belgium) for the kind gift of the Zetaprep ion exchange cartridges and housing and to Dr. J. Kadouche for the gift of the hybridoma 819 cell strain. We acknowledge the help of Dr. M.L. Fenwick for language revision of the text.
233
Reterences Caldwell, H.D., Kromhout, J. and Schachther, J. (1981) Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis.. Infect. Immun. 31, 1161. Goding, J.W. (1980) Antibody production by hybridomas. J. Immunol Methods 39, 285. Kadouche, J. Dame, C., Ketels, F., Pouletty, P., Crevat, D., Kalil, J. and Najean, Y. (1982) Analyse de diff6rentes
isoferritines par des anticorps monoclonaux. C.R. Acad. Sci. Paris 295, 443. Laemmli, U. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. Lowry, O.H., Rosebrough, N., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265. Miller, A.O.A. (1968) Etude par s6dimentation en gradient de sucrose de la ferritine marqu6e au 59Fe, extraite de cellules de HeLa. Biochim. Biophys. Acta 155, 262.
229
JIM 04349
One-step purification of mouse monoclonal antibodies by mass ion exchange chromatography on Zetaprep * F . D . M e n o z z i 1, p. V a n d e r p o o r t e n 2, C. Dejaiffe 2 and A.O.A. Miller 1 l UnitO de Biotechnologie Appliqu~e, FacultO de M~decine, Universit~ de l'Etat, Avenue du Champ de Mars, 24, 7000 Mons, and 2 Biologie et Biotechnologie S.A., Chaussbe de Binche, 101, 7000 Mons, Belgium Received 3 December 1986, revised received 21 January 1987, accepted 26 January 1987).
A rapid one-step purification procedure was developed to isolate mouse monoclonal antibodies present in the growth medium of hybridoma cultures. The procedure, based on a sulfopropyl mass ion exchange chromatography, yields considerable amounts of purified monoclonal antibody. The immunoglobulins are essentially free of transferrin and albumin, as determined by SDS-polyacrylamide gel electrophoresis. Key words: Monoclonal antibody purification; Zetaprep, sulfopropyl
Introduction Compared to monoclonal antibodies obtained from the ascitic fluid of mice injected intraperitoneally with hybridomas, which may be contaminated with various pathogens or contain proteolytic enzymes, those isolated from the growth medium used to cultivate the hybridomas in vitro - especially the synthetic ones - are less complex and therefore easier to purify. The main difficulty of this approach results from the very low concentrations at which these molecules are present and the consequent need to concentrate as well as purify them. In this work, we have purified mouse monoCorrespondence to: A.O.A. Miller, Unit~ de Biotechnologie Appliqure, Facult6 de Mrdecine, Universit~ de l'Etat, Avenue du Champ de Mars, 24, 7000 Mons, Belgium. * Work having partially benefited from the research-development project conducted with Biologie et Biotechnologie S.A. within the framework of an A.R. 123 PME agreement. Abbreviations: PBS, phosphate-buffered saline; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; SDS-Page, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SP, sulfopropyl.
clonal antibodies directly from large volumes of hybridoma culture medium, using the 2223 Zetaprep sulfopropyl mass ion exchange matrix recently developed by A M F Molecular Separations Division for LKB Produkter.
Materials and methods
Hybridomas The two hybridoma cell lines used in this work, Bambou 819 and Mompou 001 result from the polyethylene glycol 1000-mediated fusion of the SP20 murine myeloma cells with spleen cells of C3H mice immunized either with ferritin purified from HeLa cells (Miller, 1968; Kadouche et al., 1982) or with Chlamydia outer membrane complex (COMC) prepared as described by Caldwell (Caldwell et al., 1981). High titers (6-10 /~g/ml) of monoclonal antibodies (anti-MOMP antibodies) were obtained by growing the hybridomas at high cell densities in 200 cm z glass Roux flasks in supplemented D M E M containing 1% heat-inactivated newborn calf serum and 19% IBF Ultroser HY. After 2 days, the Roux
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
230 flasks were violently shaken. The medium and the cells they contain were transferred into empty Roux flasks from which the monoclonal antibody was removed following a further 24 h incubation at 37 o C. The hybridomas remaining in the initial Roux flasks were refed with fresh medium for re-use 2 days after. Sulfopropyl Zetaprep ion exchange purification of monoclonal antibodies In both of the two elution protocols which have been routinely used, the AMF Zetaprep 250 SP cartridge cast in its housing was first equilibrated with two bed volumes (500 ml) of 200 mM acetate buffer pH 5.5 at a flow rate of 300 m l / h . Then, between 300 and 450 ml of the culture supernatant from Mompou 001 hybridomas previously adjusted to pH 5.5 with HC1 were applied at a reduced flow rate of 150 m l / h (the same flow rate was maintained during all subsequent steps in both protocols) and 6 ml samples were collected. In the stepwise elution protocol, a first wash of the cartridge with 300 ml 200 mM acetate buffer p H 5.5 was followed by 250 ml of 200 mM TrisHCI pH 8.00 buffer containing successively 0, 0.2, 0.4 and 1 M NaC1. The second strategy adopted a linear 760 ml NaC1 gradient (from 0 to 0.5 M) buffered by 100 mM Tris-HC1 pH 8.00, using the LKB 11300 Ultrograd gradient mixer. All manipulations took place at room temperature. The immunoglobulin (Ig), containing fractions detected by ELISA, were pooled and an equal volume of saturated ammonium sulfate solution added in order to reach half saturation levels. After one night at 4 ° C , the precipitated IgG were collected following a 1 h centrifugation at 20000 x gay in a number 14 rotor of a preparative J-21B Beckman centrifuge run at 4°C. The Ig pellet was redissolved in 2 ml PBS, the resulting solution dialyzed for 14 h against 200 vols. of 20 mM Tris-HC1 buffer pH 7.2 and the dissolved Ig kept frozen at - 90 ° C. E L I S A detection of monoclonal antibody-containing fractions Using the method developed by Goding (Goding, 1980) the wells of 96-well PVC microtiter plates were filled with 50 #1 of a 20 # g / m l solution of COMC dissolved in PBS. Following the
usual washes and saturation of fixation sites with bovine serum albumin, the wells were filled with 50 ffl of the test solution and incubated. After supplementary washes, immobilized anti-MOMP antibodies were reacted with a 1/250 diluted alkaline phosphatase-conjugated goat anti-mouse IgG solution and their presence revealed by the addition of paranitrophenol phosphate (reading the optical density in each well at 405 nm with a Flow Titertek Multiskan). S D S gel electrophoresis - Column capacity determination - Protein determinations Analysis of the purity of the proteins eluted from the SP Zetaprep cartridge was performed by SDS-polyacrylamide gel electrophoresis on linear 4-22% gradient gels (Laemmli, 1970) using Coomassie brillant R250 to stain the separated bands. Maximum loading capacity of the column was determined by continuously charging the support with the culture supernatant until antibody molecules were detected by ELISA in the flow-through. Protein determinations were performed as described by Lowry (Lowry et al., 1951).
Results
Stepwise elution A Zetaprep ion exchange cartridge was loaded with 300 ml of Mompou 001 culture supernatant at p H 5.5 and washed with pH 5.5 buffer. It was then washed with a succession of increased concentrations of NaC1 in 200 mM Tris pH 8.00 as described in Fig. 1. The Ig (IgG1) was eluted in the first wash (zero NaC1). Polyacrylamide gel electrophoresis performed under denaturing conditions in the presence of SDS (SDS-PAGE) revealed the Ig to be contaminated by a material whose electrophoretic mobility was similar to that of albumin (Fig. 2). No contamination with transferrin was detected. Gradient elution In order to improve the purification of Ig, elution from the column was started at a lower ionic strength (100 mM Tris pH 8.00) with a continuous gradient of increasing NaC1 concentra-
231
6~.
E
E=.
Ex
E=
/2
l: I t
' "
°o
'°!
i
.... 5 25
50
75
I00
125
Fig. 1. Stepwise elution profile of 300 ml Mompou 001 supernatant on SP Zetaprep 250. The protein elution was monitored by UV recording at 280 nm (O O) and the antibody elution was determined by a specific ELISA (O O).
150
FRACTION NUMBER (6ml)
1
2
3
4
tion. With this technique, the anti-MOMP Ig was eluted at 250 mM NaC1 (Fig. 3). SDS-PAGE analysis (Fig. 2) of the Ig eluted in this way showed only two bands corresponding to the heavy and fight chains. Neither albumin nor transferrin could be detected. Table I summarizes the salient characteristics of this mode of elution.
5
6
7
W
N
17 Fig. 2. SDS-PAGE analysis of the fractions eluted from the SP-Zetaprep. Lane I: Mompou 001 culture supematant. Lane 2: Bovine serum albumin. Lane 3: Mompou 001 monoclonal antibodies obtained by stepwise elution from three independent experiments (as in lane 5), pooled and submitted to a new stepwise elution chromatography on SP Zetaprep. Little albumin contamination is visible. Lane 4: Fractions 129 to 142 from the gradient elution of 300 ml Bambou 819 culture supernatant. Lane 5: Fractions 90-96 from the stepwise elution of 300 ml Mompou 001 culture supernatant. Lane 6: Fractions 130-139 from the gradient elution of 300 ml Mompou 001 culture supernatant. Lane 7: Molecular weight markers: phosphorylase B (94 KD), albumin (67 KD), ovalbumin (43 KD), carbonic anhydrase (30 KD), heavy and light ferritin sub-units (19 and 17 KD).
232 TABLE I C O M P A R A T I V E S U M M A R Y O F T H E T W O M O N O C L O N A L A N T I B O D Y P U R I F I C A T I O N S ON SP Z E T A P R E P 250 F R O M IN VITRO C U L T U R E S U P E R N A T A N T S OF T H E B A M B O U 819 A N D M O M P O U 001 H Y B R I D O M A S ( G R A D I E N T SALT ELUTION) Cell line
Monoclonal IgG secreted (/~g/ml)
Hybridoma supernatant loaded on the SP Zetaprep (ml)
Monoclonal antibody recovery (%)
Purification ~ factor
Concentration b factor
Bambou 819 clone M o m p o u 001 clone
8 6
300 300
89 90
15 10
3 3
Purification factor: calculated from the relation A z / A o × B o / B z where A z and A o are the mean concentration ( ~ g / m l ) of M o m p o u IgG in the eluate from the Zetaprep ( A z ) and in the culture supernatant (Ao), B z and Bo being the mean protein concentration (t*g/ml) in the eluate from the Zetaprep ( B z ) and in the culture supernatant (Bo). b Concentration factor: calculated as the ratio of the volume of culture supernatant put on the Zetaprep cartridge divided by the volume of the eluate containing the IgG. a
25
50
75
IN
12S
150
175
FRACTION NUMBER (6ml)
Fig. 3. Gradient elution profile of 300 ml M o m p o u 001 supernatant on SP Zetaprep 250. A linear gradient salt from 0 to 500 m M NaC1 was used to remove the proteins adsorbed to the matrix ( I m). The protein (O O) and monoclonal antibody (O O) elutions were monitored as in Fig. 1. The flow rate was maintained constant at 150 m l / h and the cartridge developed as described in the materials and methods section.
Tris pH 8.0, 1 M NaC1 and 4 bed volumes of 200 m M acetate buffer pH 5.5) takes approximately the same time, as many as two successive cycles of purification and regeneration can be performed per 24 h allowing as much as 3 mg of pure monoclonal antibody to be obtained daily. Extension of this technology to the purification of IgG1 molecules, secreted by the murine Bambou clone 819 against ferritin extracted from HeLa ceils, gave identical results (except for the elution of the antibody at 260 mM NaC1). Gradient elution is not as well suited as stepwise elution to operations on an industrial scale. Our results show that stepwise elution yields Ig contaminated by albumin (present in the newborn calf serum added to the growth medium). It is possible that careful optimization of the latter procedure could eliminate the observed contamination. Alternatively, the high flow rate of the Zetaprep cartridge could be used for the large scale removal of albumin by a stepwise elution protocol on a SP Zetaprep 250 followed downstream by a Protein A-Zetaprep cartridge.
Discussion
Acknowledgements
SP Zetaprep ion exchange chromatography allows easy, rapid and almost quantitative recovery of murine monoclonal antibodies produced by hybridomas grown in dilute serum medium. As much as 500 ml of Ig-containing growth medium can be passed through the cartridge in less than 5 h. Because regeneration (1 bed volume of 100 mM
We are much indebted to Mr. J. Jansseune (LKB, Belgium) for the kind gift of the Zetaprep ion exchange cartridges and housing and to Dr. J. Kadouche for the gift of the hybridoma 819 cell strain. We acknowledge the help of Dr. M.L. Fenwick for language revision of the text.
233
Reterences Caldwell, H.D., Kromhout, J. and Schachther, J. (1981) Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis.. Infect. Immun. 31, 1161. Goding, J.W. (1980) Antibody production by hybridomas. J. Immunol Methods 39, 285. Kadouche, J. Dame, C., Ketels, F., Pouletty, P., Crevat, D., Kalil, J. and Najean, Y. (1982) Analyse de diff6rentes
isoferritines par des anticorps monoclonaux. C.R. Acad. Sci. Paris 295, 443. Laemmli, U. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. Lowry, O.H., Rosebrough, N., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265. Miller, A.O.A. (1968) Etude par s6dimentation en gradient de sucrose de la ferritine marqu6e au 59Fe, extraite de cellules de HeLa. Biochim. Biophys. Acta 155, 262.