Microbial Pathogenesis 1991 ; 10: 4 1 1 -417
Neutralizing monoclonal antibodies to Pasteurella haemolytica leukotoxin affinity-purify the toxin from crude culture supernatants Martha J . Gentry* and S . Srikumarant Department of Veterinary Science, University of Nebraska, Lincoln, Nebraska 68583-0905, U.S .A .
(Received August 27, 1990 ; accepted in revised form February 11, 1991)
Gentry, M . J . (Dept of Veterinary Science, University of Nebraska, Lincoln, Nebraska 685830905, U .S .A .) and S . Srikumaran . Neutralizing monoclonal antibodies to Pasteurella haemolytica leukotoxin affinity-purify the toxin from crude culture supernatants . Microbial Pathogenesis 1991 ; 10 : 411-417 .
The leukotoxin of Pasteurella haemolytica is a major virulence factor of the organism . It is an unstable protein which has proven very difficult to purify using traditional techniques . Hybridomas secreting monoclonal antibodies (mAbs) to P. haemolytica leukotoxin were derived from spleen cells of a mouse immunized with crude culture supernatant . Five hybridomas secreting mAbs specific for the leukotoxin were stabilized . Each of the mAbs reacted with a protein of approximately 100 kDa in toxic culture supernatants, and two of them completely neutralized the toxin in vitro . Affinity chromatography of crude culture supernatant on a column prepared with one of the neutralizing mAbs resulted in the isolation of biologically active toxin . Key words : Pasteurella haemolytica ; leukotoxin neutralization ; affinity- purification ; monoclonal
antibody .
Introduction Pasteurella haemolytica, the etiological agent of bovine pneumonic pasteurellosis,
produces a leukotoxin which is excreted into the medium during logarithmic-phase growth of the organism in vitro .' This toxin is specific for ruminant leukocytes,"' and has been suggested to be a major virulence factor of the organism responsible for leukocyte damage in the lung .' , ' Leukotoxin-induced neutrophil lysis and degranulation have been implicated as the primary cause of the acute inflammation which is characteristic of pneumonic pasteurellosis .' Progress has been slow in determining the biochemical nature and even the molecular mass of the toxin because of difficulties encountered in its purification . Traditional techniques such as ultrafiltration,' gel filtration,' and column chromatography9 have produced only partially purified materials . Cloning of the leukotoxin structural gene into Escherichia coli by two groups of investigators resulted in expression of 102 kDa and 105 kDa proteins, respectively .' 011 " Present address : Research Service 151, Department of Veterans Affairs Medical Center, 4101 Woolworth Avenue, Omaha, Nebraska 68105, U .S .A . t Author to whom correspondence should be addressed .
0882-4010/91 /050411 +07 503 .00/0
© 1991 Academic Press Limited
41 2
M . J . Gentry and S . Srikumaran
The unavailability of purified P. haemolytica leukotoxin has made attempts to produce leukotoxin-specific antibodies more difficult . The purpose of the present study was to develop mAbs against the leukotoxin and use them to affinity-purify active toxin from crude culture supernatants . To circumvent the problem of the unavailability of purified leukotoxin for the screening of hybridomas, a functional neutralization assay rather than a binding assay was used .
Results Hybridoma production and testing Five hybridomas (MM601-MM605) secreting mAbs to P. haemolytica leukotoxin were produced by fusion of the murine cell line SP-2/0 with spleen cells from a mouse immunized with toxin produced in medium supplemented with mouse serum (MStoxin) . The Ig isotypes, and the neutralization and radioimmunoassay (RIA) titers of the mAbs secreted by these hybridomas are given in Table 1 . The hybridomas designated MM601 and MM602 were derived from the only two wells out of 938 tested which contained culture fluids that could effectively neutralize P. haemolytica leukotoxin in a 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye reduction neutralization assay . Hybridomas MM603, MM604, and MM605 initially yielded culture fluids that could partially neutralize the toxin . After subcloning, however, their mAbs could no longer neutralize the toxin even though they could bind to it as demonstrated by RIA . Each of the mAbs recognized the same protein band of approximately 100 kDa on immunoblots of crude toxin prepared in medium supplemented with bovine serum albumin (BSA-toxin) . An additional faint band was recognized in BSA-toxin at a molecular weight of approximately 97 kDa . Affinity purification of the toxin mAb MM601 was used for affinity purification of the toxin . Pools of the leukotoxin fractions eluted from the affinity column were toxic for bovine leukocytes in the MTT assay (Table 2) . During toxin production in BSA-supplemented medium, the growth and subsequent removal of P. haemolytica organisms did not result in an increase of total protein in the culture medium (Table 2) . The amount of actual toxin protein contained in the crude culture supernatant, therefore, could not be determined . Concentration of the crude toxin resulted in an 8 .5-fold increase in protein concentration and a greater than 10-fold increase in its toxic units/ml . The concentrated toxin loaded onto the affinity column contained 8 .5 mg/mI of protein, 1 .19 mg/mI of
Table 1
Monoclonal antibodies to P. haemolytica leukotoxin Neutralization titer'
RIA titer b
Clone
Isotype
Culture fluid
Ascites fluid
Culture fluid
Ascites fluid
MM601 MM602 MM603 MM604 MM605
IgG1 IgG1 IgG215 IgG2b IgG2a
32 32
Neutralizing monoclonal antibodies to Pasteurella haemolytica leukotoxin affinity-purify the toxin from crude culture supernatants Martha J . Gentry* and S . Srikumarant Department of Veterinary Science, University of Nebraska, Lincoln, Nebraska 68583-0905, U.S .A .
(Received August 27, 1990 ; accepted in revised form February 11, 1991)
Gentry, M . J . (Dept of Veterinary Science, University of Nebraska, Lincoln, Nebraska 685830905, U .S .A .) and S . Srikumaran . Neutralizing monoclonal antibodies to Pasteurella haemolytica leukotoxin affinity-purify the toxin from crude culture supernatants . Microbial Pathogenesis 1991 ; 10 : 411-417 .
The leukotoxin of Pasteurella haemolytica is a major virulence factor of the organism . It is an unstable protein which has proven very difficult to purify using traditional techniques . Hybridomas secreting monoclonal antibodies (mAbs) to P. haemolytica leukotoxin were derived from spleen cells of a mouse immunized with crude culture supernatant . Five hybridomas secreting mAbs specific for the leukotoxin were stabilized . Each of the mAbs reacted with a protein of approximately 100 kDa in toxic culture supernatants, and two of them completely neutralized the toxin in vitro . Affinity chromatography of crude culture supernatant on a column prepared with one of the neutralizing mAbs resulted in the isolation of biologically active toxin . Key words : Pasteurella haemolytica ; leukotoxin neutralization ; affinity- purification ; monoclonal
antibody .
Introduction Pasteurella haemolytica, the etiological agent of bovine pneumonic pasteurellosis,
produces a leukotoxin which is excreted into the medium during logarithmic-phase growth of the organism in vitro .' This toxin is specific for ruminant leukocytes,"' and has been suggested to be a major virulence factor of the organism responsible for leukocyte damage in the lung .' , ' Leukotoxin-induced neutrophil lysis and degranulation have been implicated as the primary cause of the acute inflammation which is characteristic of pneumonic pasteurellosis .' Progress has been slow in determining the biochemical nature and even the molecular mass of the toxin because of difficulties encountered in its purification . Traditional techniques such as ultrafiltration,' gel filtration,' and column chromatography9 have produced only partially purified materials . Cloning of the leukotoxin structural gene into Escherichia coli by two groups of investigators resulted in expression of 102 kDa and 105 kDa proteins, respectively .' 011 " Present address : Research Service 151, Department of Veterans Affairs Medical Center, 4101 Woolworth Avenue, Omaha, Nebraska 68105, U .S .A . t Author to whom correspondence should be addressed .
0882-4010/91 /050411 +07 503 .00/0
© 1991 Academic Press Limited
41 2
M . J . Gentry and S . Srikumaran
The unavailability of purified P. haemolytica leukotoxin has made attempts to produce leukotoxin-specific antibodies more difficult . The purpose of the present study was to develop mAbs against the leukotoxin and use them to affinity-purify active toxin from crude culture supernatants . To circumvent the problem of the unavailability of purified leukotoxin for the screening of hybridomas, a functional neutralization assay rather than a binding assay was used .
Results Hybridoma production and testing Five hybridomas (MM601-MM605) secreting mAbs to P. haemolytica leukotoxin were produced by fusion of the murine cell line SP-2/0 with spleen cells from a mouse immunized with toxin produced in medium supplemented with mouse serum (MStoxin) . The Ig isotypes, and the neutralization and radioimmunoassay (RIA) titers of the mAbs secreted by these hybridomas are given in Table 1 . The hybridomas designated MM601 and MM602 were derived from the only two wells out of 938 tested which contained culture fluids that could effectively neutralize P. haemolytica leukotoxin in a 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye reduction neutralization assay . Hybridomas MM603, MM604, and MM605 initially yielded culture fluids that could partially neutralize the toxin . After subcloning, however, their mAbs could no longer neutralize the toxin even though they could bind to it as demonstrated by RIA . Each of the mAbs recognized the same protein band of approximately 100 kDa on immunoblots of crude toxin prepared in medium supplemented with bovine serum albumin (BSA-toxin) . An additional faint band was recognized in BSA-toxin at a molecular weight of approximately 97 kDa . Affinity purification of the toxin mAb MM601 was used for affinity purification of the toxin . Pools of the leukotoxin fractions eluted from the affinity column were toxic for bovine leukocytes in the MTT assay (Table 2) . During toxin production in BSA-supplemented medium, the growth and subsequent removal of P. haemolytica organisms did not result in an increase of total protein in the culture medium (Table 2) . The amount of actual toxin protein contained in the crude culture supernatant, therefore, could not be determined . Concentration of the crude toxin resulted in an 8 .5-fold increase in protein concentration and a greater than 10-fold increase in its toxic units/ml . The concentrated toxin loaded onto the affinity column contained 8 .5 mg/mI of protein, 1 .19 mg/mI of
Table 1
Monoclonal antibodies to P. haemolytica leukotoxin Neutralization titer'
RIA titer b
Clone
Isotype
Culture fluid
Ascites fluid
Culture fluid
Ascites fluid
MM601 MM602 MM603 MM604 MM605
IgG1 IgG1 IgG215 IgG2b IgG2a
32 32
