RESEARCH
NOTE
A STRATEGY FOR PRODUCTION OF MONOCLONAL ANTIBODIES TO E~~~~~~~~~US G~A~U~~SUS ANTIGEN 5 AND ANTIGEN B D. LIU,*~ M. D. RICKARD~
and M. W. LIGHTOWLERS*
*University of Melbourne, Veterinary Clinical Centre, Princes Highway, $Z.S.I.R.O.
Division
of Animal
Health,
Parkviile,
Werribee, Victoria Victoria 3052, Australia
(Received 12 June 1992; accepted 15 August
3030, Australia
1992)
Abstract-Ln D., RICKARD M. D. and LIGHTOWLERS M. W. 1992. A strategy for production of monoclonal antibodies to Echinococcus granulosus Antigen 5 and Antigen B. International Journal,for Parasitology 22: 1013-1016. Serum antibody responses to sheep hydatid cyst thud (SHCF) and a purified Antigen 5 (Ag5) were examined in ELISA, immunoelectrophoresis (IEP) and immunoprecipitation (IP) to facilitate production of monoclonal antibodies (MAb) to E. gr~~nul~~u~~ Ag5 and Antigen B (AgB). Although sera from mice immunized with SHCF contained antibodies of various classes, the fusions using these donor mice resulted in mainly anti-AgB MAb, possibly due to the preferential selection of MAb to AgB by the SHCF-based ELISA screening system. Donor mice immunized with Ag5 also produced several classes of antibodies, and the resultant fusions enabled selection of IgG MAb to Ag5. INDEX
KEY WORDS:
Echi?~ococcusgranulosus; Antigen
M~NOCLONAL antibodies (MAb) have proved to be useful tools for analysis of bacterial, viral and parasitic antigens and for improved diagnosis of their infections in man and animals. In the case of the unilocular hydatid parasite Echinococcus grunulosus, recent studies have shown that when using MAb to the major hydatid antigens, Antigen 5 (Ag5) and Antigen B (AgB), the antigenic components and subunits of E. gvunulosus could be more clearly defined, and the specific detection of human hydatidosis enhanced (Di Felice, Pini, Afferni & Vicari, 1986; Lightowlers, Liu, Haralambous & Rickard, 1989; Liu, Lightowlers & Rickard, 1992a). In the process of generating MAb to E. granulosus Ag5 and AgB, we utilized spleen cells from donor mice sensitized with sheep hydatid cyst fluid (SHCF) and a SHCF-based ELISA screening system, and obtajned anti-AgB MAb which formed the typical AgB band with SHCF in immunoelectrophoresis (IEP) (Lightowlers et al., 1989). However, we experienced some unexpected difficulty in producing anti-Ag5 MAb which was able to form the classical Arc 5 band with SHCF in IEP using the same
t Present address and address for all correspondence: Department of Food and Agriculture, Pastoral and Veterinary Institute, Private Bag 105, Hamilton, Victoria 3300, Australia.
5; Antigen
B; monoclonal antibody.
procedures. To find out if this was due to a lack of IgG antibody response in SHCF-sensitized mice, we conducted a study on the antibody responses in mice immunized with SHCF. In addition, an alternative strategy was taken to use a purified Ag5 for preparation of donor mice for anti-Ag5 MAb production. Here we report our observations on the antibody classes in sera of mice immunized with SHCF and Ag5, and describe the outcome of fusions using spieen cells from these donor mice and myeloma cells. Sheep hydatid cyst fluid (SHCF) was prepared as previously described (Liu et al., 1992~1)and Antigen 5 (Ag5) was purified from SHCF using a HPLC Protein Pak 300 column (Waters) (Liu, Lightowlers & Rickard. 1992b). BALBjc mice of both sexes at 8-10 weeks of age were each injected S.C. and i.p. with approximately 100 pg SHCF antigen in 100 ,~l mouse tonicity PBS (MTPBS) emulsified with 100 ~1 Freund’s complete adjuvant (Difco). The same dose of antigen emulsified with 100 ~1 Freund’s incomplete adjuvant was injected S.C. and i.p. 2, 4 and 6 weeks after the first injection. Blood samples were taken from the mice at the 7th week and antibody responses were assessed in ELISA and in TEP to determine their suitability as spleen donors for MAb production. Immunization of mice using HPLC-purified Ag.5 1013
D. Lw, M. D. RICKARD
I014
and M. W.
LICHTOWLERS
SHCF + MxSHCF
Ag5 + MxAg5
0.15
0.15
0.25
0.25
0.35
0.35
z 2
0.45
0.45 1
2
4
8
16
32
64
128
256
SHCF + NMS
Ag5 + NMS
0.45
0.45 1
2
4
8
Serum
16
dilution
32
64
128
256
(-2)
1
2
4
8
Serum
16
dilution
32
64
128
256
(-2)
FIG. I. Examination
of antibody classes in sera from mice immunized with SHCF and Ag5 in ELISA. Rabbit anti-mouse IgG,, IgA and IgM and urease-labelled sheep anti-rabbit IgG conjugate were used. For IgG,, IgG2,, IgG,,, SHCF + MxSHCF and SHCF + NMS, SHCF at 10 pg ml ’, and for Ag5+MxAgS and Ag5+NMS, Ag5 also at IO ug ml-’ were added. Mouse sera were tested at 13100-l/25,600 dilutions. The urease substrate reaction was read at an absorbance of 450 nm. The heading SHCF + MxSHCF indicates anti-SHCF antibodies in sera from a SHCF-immunized mouse detected by rabbit anti-mouse IgG, (m), IgG,, (u), IgG,, (I& IgG, (a), IgA (0) and IgM (B). The heading Ag5 + MxAgS indicates anti-Ag5 antibodies in sera from an AgS-immunized mouse detected by rabbit anti-mouse IgG, (a). IgG,, (a), IgG,, (a), IgG, ( q ), IgA (a) and IgM (m). The headings SHCF + NMS and Ag5 + NMS indicate normal mouse serum controls for SHCF and Ag5 in ELISA.
was carried out exactly as described for using SHCF except that an antigen dose of approximately 50 pg per mouse was used for each injection. In addition, one group of immunized mice was injected in the 4th and 6th weeks with 50 ,ug Ag5 plus 50 fig keyhole limpet haemocyanin (KLH, Sigma) in 200 ~1 MTPBS to enhance antibody responses. Sera from immunized mice were tested in ELISA and IEP using Ag.5 and normal sheep serum (NSS) to confirm the specificity of anti-Ag5 antibodies. Mice were boosted iv. and i.p. with 500 gig SHCF in 500 ~1 MTPBS 3 days prior to fusion. The standard monoclonal antibody procedures (Galfre & Milstein, 1981) were used for production of MAb to E. granulosus Ag5 and AgB. The resultant hybrid supernatants were screened against SHCF and NSS in ELISA. Clones showing SHCFpositive and NSS-negative ELISA were selected and subcloned at least twice. The potentially useful hybridomas were propagated in pristane-primed mice for ascites fluid production, and MAb were partially the characterized by examining
precipitation arcs formed between hybridoma ascites fluid and SHCF in IEP. Enzyme-linked immunosorbent assay (ELISA) was similar to that used in Liu c)t nl. (1992af. To screen hybridoma cultures, SHCF and NSS at 10 ,q ml-’ (total protein) and undiluted MAb supernatants were added. This was followed by urease-labelled sheep anti-mouse Ig or urease-labelled sheep antimouse IgG (CSL, Australia) in the later experiments. To examine the antibody classes; SHCF or Ag5 at 10 t_lg ml-’ and sera from SHCF- or Ag5-immunized mice were added, which was followed by rabbit antimouse IgG,, IgG>,, IgG,,, IgG,, IgA and IgM (Miles), and then urease-labelled sheep anti-rabbit IgG (CSL, Australia). The assay was developed using urease substrate and read at an absorbance of either 450 or 590 nm. Immunoelectrophoresis (IEP) of MAb ascites and mouse antisera to SHCF and Ag5 was performed as in Liu et d. (1992b). Immunoprecipitation (IP) analysis was carried out as described in Lightowlers ff al. (1989). Immunization with SHCF resulted in the production
Research Note of all major classes and subclasses of anti-hydatid antibodies in mice. A typical pattern of IgG,, IgG,,, IgG,,, IgG,, IgA and IgM antibodies is shown in Fig. 1 SHCF+ MxSHCF. Anti-hydatid IgG, and IgM antibodies were detected in ELISA at I/12,800 and I/ 800 serum dilutions and specific IgG2,, IgG,,, IgG, and IgA were present at l/40&1/800 dilutions from this particular mouse. Anti-AgS IgG antibodies were clearly present in sera of mice immunized with SHCF as these sera reacted strongly with the purified Ag5 in ELISA using a urease-labelled sheep anti-mouse IgG conjugate (CSL, Australia) (data not shown). As expected, sera from SHCF-immunized mice contained antibodies to all the major antigens of E. granulosus such as Ag5 and AgB when examined in IEP (data not shown). Immunoprecipitation analysis of mouse sera to SHCF indicated that these sera recognized various components of SHCF including those of Ag5 (24 and 38 kDa) and of AgB (8, 16 and 24 kDa) as determined in Lightowlers et al. (1989). Ag5 also stimulated substantial antibody responses in immunized mice (Fig. 1 Ag5 + MxAg5). Specific IgG, and IgM antibodies were detected at serum dilutions of l/3200 and l/400 from this particular mouse. Some IgG2, and IgGzh antibodies were also present. Mice immunized with Ag5 plus KLH showed a similar, but enhanced antibody response to Ag5 compared with mice immunized with Ag5 with initial Freund’s complete adjuvant only. IgG,, IgG,, and IgM antibodies were particularly noticeable, but IgGzb and IgGi antibodies were also produced (data not shown). Sera from non-immunized control mice did not have any detectable antibodies to hydatid antigens in both ELISA and IEP (data not shown), but they as well as SP2 ascites did precipitate a band of 51 kDa in immunoprecipitation which was probably the same as the band of 55 kDa reported by Shepherd and McManus (1987) which was postulated as sheep host heavy chain immunoglobulin. Sera of mice immunized with HPLC-purified Ag5 formed only an Arc 5 with SHCF (Liu et al., 1992b), and did not show any precipitating band with NSS in IEP (data not shown), although the presence of sheep serum components was confirmed in ELISA using Ag5 vs rabbit antiserum to NSS and NSS vs mouse antisera to Ag5 (data not shown). Mouse sera to Ag.5 precipitated bands in immunoprecipitation identical to those recognized by anti-Ag5 MAb (Lightowlers et al., 1989). Two hybrid cell lines were selected as a result of fusions between SP2 or NSI and spleen cells from SHCF-immunized mice. They were 31.15, which secretes IgGzh antibodies to AgB, and 39B3, which secretes IgG,, antibodies to AgB. Ascites fluid from
1015
these two MAb formed with SHCF an AgB-type arc in IEP and an AgB band pattern in immunoprecipitation (Lightowlers et al., 1989), and did not react with NSS in IEP (data not shown). Several IgG MAb to AgB that formed no immunoelectrophoretic bands were also produced. The identity of these MAb was determined in immunoprecipitation by their formation of a band pattern characteristic of anti-AgB MAb (Lightowlers et al., 1989). In addition, a large number of hybrid clones producing specific IgM antibodies were also obtained from the fusions between myeloma cells and spleen cells from SHCF-sensitized mice. Since anti-hydatid IgM MAb tended to form morphologically uncharacteristic bands in IEP with SHCF (data not shown), they were not pursued further in the later experiments. Despite the evidence of anti-Ag5 IgG antibodies in sera of mice immunized with SHCF, specific IgG MAb to Ag5 were not derived from these fusions. On the other hand, using donor mice immunized with Ag5, one hybrid cell line (24.14) which secretes IgG,, antibodies to Ag5 was produced. A second clone (6lAl2) which secretes IgGzh antibodies was derived from a fusion using spleen cells from Ag5and KLH-immunized mice. Ascites from both clones formed a typical Arc 5 with SHCF in IEP and an band in pattern immunoprecipitation Ag5 (Lightowlers et al., 1989). None of them showed any IEP band with NSS (data not shown). Further, a number of anti-Ag5 IgG MAb that showed no band in IEP were selected. Again, their identity was determined in immunoprecipitation by their formation of a band pattern characteristic of antiAg5 MAb (Lightowlers et al., 1989). A whole spectrum of specific antibody subclasses was detected in sera of mice injected with SHCF, especially of IgG, and IgM. Although IgG antibodies to Ag5 were present in sera from these mice in ELISA, IgG MAb to Ag5 were not obtained. It was possible that MAb to AgB might have been preferentially selected using a SHCF-based ELISA for screening hybrid cells because of stronger signals resulting from the higher proportion of AgB to Ag5 (10 to 1) in SHCF (Musiani, Piantelli, Lauriola. Arru & Pozzouli, 1978). Alternatively, it might be due to the possibility that lower numbers of anti-Ag5 B cells were induced in mice immunized with SHCF compared with those of anti-AgB B cells. It appeared that in order to facilitate the production of IgG MAb to Ag5, it might be necessary to employ a purified Ag5 to either immunize spleen donor mice or screen hybrid supernatants from fusions using spleen cells from SHCF-sensitized mice. Ag5 purified by HPLC was highly immunogenic in that it stimulated production of several different
1016
D. LIU, M. D. RKKARD and M. W. LIGHTOWLERS
antibody classes in sera of immunized mice. KLH enhanced the antibody response in AgS-sensitized mice. Sera from mice immunized with Ag5 formed only one Arc 5 band with SHCF and did not react with NSS in IEP, although mouse sera to Ag5 reacted with NSS in the more sensitive ELISA. It seemed that mice displayed a preferential immune response to Ag5 over other minor elements of hydatid antigens and sheep serum components present in HPLC-purified Ag5 preparation as assessed in IEP (Liu et al., 1992b). As a result of fusions between myelomas and spleen cells from AgS-sensitized mice, at least two IgG MAb to Ag5 were generated even though the same screening system was used. It is also of interest to observe that ascites from almost all IgM secreting hybrid cells but only some IgG secreting cells readily formed precipitating bands in IEP. Perhaps this reflects the fact that the nature of IgM molecules favours the formation of precipitation bands in comparison with that of IgG molecules. Although some of the IgG MAb failed to form any precipitation bands with SHCF in IEP, they did show similar band patterns to those formed by anti-Ag5 and AgB MAb in immunoprecipitation (Lightowlers et al., 1989). This suggests that for confirmation
of MAb
to
the
major
antigens
of E.
be a more sensitive IEP. assay than However, the demonstration of classical Arc 5 and AgB bands in IEP is an ultimate criterion for selection of MAb to Ag5 and AgB.
granulosus,
immunoprecipitation
might
Acknowledgements-This study was supported from the University of Melbourne Veterinary Fund and the National Health and Medical Council of Australia.
by grants Research Research
REFERENCES DI FELICE G., PINI C., AFFERNI C. & VICARI G. 1986. Purification and partial characterization of the major antigen of Echinococcus granulosus (antigen 5) with monoclonal antibodies. Molecular and Biochemical Parasitology 20: 133- 142. GALFRE G. & MILSTEINC. 1981. Preparation of monoclonal antibodies: strategies and procedures. Methodr in Enzymology 73: 146. L~GHTOWLERSM. W., LIU D., HARALAMBOUSA. & RICKAR~ M. D. 1989. Subunit composition and specificity of the major cyst fluid antigens of Echinococcus granulosus. Molecular and Biochemical Parasitology 37: I7 I-1 82. LIU D., LICHTOWLERS M. W. & RICKARD M. D. 1992a. Evaluation of a monoclonal antibody based competition ELISA for diagnosis of human hydatidosis. Para.sito/og~~ 104: 357-361. LIU D., LICHTOWLERS M. W. & RICKARD M. D. 1992b. Examination of murine antibody response to secondary hydatidosis using ELISA and immunoelectrophoresis. Parasile Immunology 14: 239-248. MUSIANI P., PIANTELLI M.. LAURIOLA L., ARR~J E. & POZZOULI R. 1978. Echinococcxv granulosus: specitic quantification of the most immunoreactive antigens in hydatid fluids. Journal qf Clinical Pathology 31: 475%47X. SHEPHERD J. C. & MCMANUS D. P. 1987. Specific and crossreactive antigens of Echinococcus granulosus hydatid cyst fluid. Molecular and Biochemical Parasitology. 25: 143154.
NOTE
A STRATEGY FOR PRODUCTION OF MONOCLONAL ANTIBODIES TO E~~~~~~~~~US G~A~U~~SUS ANTIGEN 5 AND ANTIGEN B D. LIU,*~ M. D. RICKARD~
and M. W. LIGHTOWLERS*
*University of Melbourne, Veterinary Clinical Centre, Princes Highway, $Z.S.I.R.O.
Division
of Animal
Health,
Parkviile,
Werribee, Victoria Victoria 3052, Australia
(Received 12 June 1992; accepted 15 August
3030, Australia
1992)
Abstract-Ln D., RICKARD M. D. and LIGHTOWLERS M. W. 1992. A strategy for production of monoclonal antibodies to Echinococcus granulosus Antigen 5 and Antigen B. International Journal,for Parasitology 22: 1013-1016. Serum antibody responses to sheep hydatid cyst thud (SHCF) and a purified Antigen 5 (Ag5) were examined in ELISA, immunoelectrophoresis (IEP) and immunoprecipitation (IP) to facilitate production of monoclonal antibodies (MAb) to E. gr~~nul~~u~~ Ag5 and Antigen B (AgB). Although sera from mice immunized with SHCF contained antibodies of various classes, the fusions using these donor mice resulted in mainly anti-AgB MAb, possibly due to the preferential selection of MAb to AgB by the SHCF-based ELISA screening system. Donor mice immunized with Ag5 also produced several classes of antibodies, and the resultant fusions enabled selection of IgG MAb to Ag5. INDEX
KEY WORDS:
Echi?~ococcusgranulosus; Antigen
M~NOCLONAL antibodies (MAb) have proved to be useful tools for analysis of bacterial, viral and parasitic antigens and for improved diagnosis of their infections in man and animals. In the case of the unilocular hydatid parasite Echinococcus grunulosus, recent studies have shown that when using MAb to the major hydatid antigens, Antigen 5 (Ag5) and Antigen B (AgB), the antigenic components and subunits of E. gvunulosus could be more clearly defined, and the specific detection of human hydatidosis enhanced (Di Felice, Pini, Afferni & Vicari, 1986; Lightowlers, Liu, Haralambous & Rickard, 1989; Liu, Lightowlers & Rickard, 1992a). In the process of generating MAb to E. granulosus Ag5 and AgB, we utilized spleen cells from donor mice sensitized with sheep hydatid cyst fluid (SHCF) and a SHCF-based ELISA screening system, and obtajned anti-AgB MAb which formed the typical AgB band with SHCF in immunoelectrophoresis (IEP) (Lightowlers et al., 1989). However, we experienced some unexpected difficulty in producing anti-Ag5 MAb which was able to form the classical Arc 5 band with SHCF in IEP using the same
t Present address and address for all correspondence: Department of Food and Agriculture, Pastoral and Veterinary Institute, Private Bag 105, Hamilton, Victoria 3300, Australia.
5; Antigen
B; monoclonal antibody.
procedures. To find out if this was due to a lack of IgG antibody response in SHCF-sensitized mice, we conducted a study on the antibody responses in mice immunized with SHCF. In addition, an alternative strategy was taken to use a purified Ag5 for preparation of donor mice for anti-Ag5 MAb production. Here we report our observations on the antibody classes in sera of mice immunized with SHCF and Ag5, and describe the outcome of fusions using spieen cells from these donor mice and myeloma cells. Sheep hydatid cyst fluid (SHCF) was prepared as previously described (Liu et al., 1992~1)and Antigen 5 (Ag5) was purified from SHCF using a HPLC Protein Pak 300 column (Waters) (Liu, Lightowlers & Rickard. 1992b). BALBjc mice of both sexes at 8-10 weeks of age were each injected S.C. and i.p. with approximately 100 pg SHCF antigen in 100 ,~l mouse tonicity PBS (MTPBS) emulsified with 100 ~1 Freund’s complete adjuvant (Difco). The same dose of antigen emulsified with 100 ~1 Freund’s incomplete adjuvant was injected S.C. and i.p. 2, 4 and 6 weeks after the first injection. Blood samples were taken from the mice at the 7th week and antibody responses were assessed in ELISA and in TEP to determine their suitability as spleen donors for MAb production. Immunization of mice using HPLC-purified Ag.5 1013
D. Lw, M. D. RICKARD
I014
and M. W.
LICHTOWLERS
SHCF + MxSHCF
Ag5 + MxAg5
0.15
0.15
0.25
0.25
0.35
0.35
z 2
0.45
0.45 1
2
4
8
16
32
64
128
256
SHCF + NMS
Ag5 + NMS
0.45
0.45 1
2
4
8
Serum
16
dilution
32
64
128
256
(-2)
1
2
4
8
Serum
16
dilution
32
64
128
256
(-2)
FIG. I. Examination
of antibody classes in sera from mice immunized with SHCF and Ag5 in ELISA. Rabbit anti-mouse IgG,, IgA and IgM and urease-labelled sheep anti-rabbit IgG conjugate were used. For IgG,, IgG2,, IgG,,, SHCF + MxSHCF and SHCF + NMS, SHCF at 10 pg ml ’, and for Ag5+MxAgS and Ag5+NMS, Ag5 also at IO ug ml-’ were added. Mouse sera were tested at 13100-l/25,600 dilutions. The urease substrate reaction was read at an absorbance of 450 nm. The heading SHCF + MxSHCF indicates anti-SHCF antibodies in sera from a SHCF-immunized mouse detected by rabbit anti-mouse IgG, (m), IgG,, (u), IgG,, (I& IgG, (a), IgA (0) and IgM (B). The heading Ag5 + MxAgS indicates anti-Ag5 antibodies in sera from an AgS-immunized mouse detected by rabbit anti-mouse IgG, (a). IgG,, (a), IgG,, (a), IgG, ( q ), IgA (a) and IgM (m). The headings SHCF + NMS and Ag5 + NMS indicate normal mouse serum controls for SHCF and Ag5 in ELISA.
was carried out exactly as described for using SHCF except that an antigen dose of approximately 50 pg per mouse was used for each injection. In addition, one group of immunized mice was injected in the 4th and 6th weeks with 50 ,ug Ag5 plus 50 fig keyhole limpet haemocyanin (KLH, Sigma) in 200 ~1 MTPBS to enhance antibody responses. Sera from immunized mice were tested in ELISA and IEP using Ag.5 and normal sheep serum (NSS) to confirm the specificity of anti-Ag5 antibodies. Mice were boosted iv. and i.p. with 500 gig SHCF in 500 ~1 MTPBS 3 days prior to fusion. The standard monoclonal antibody procedures (Galfre & Milstein, 1981) were used for production of MAb to E. granulosus Ag5 and AgB. The resultant hybrid supernatants were screened against SHCF and NSS in ELISA. Clones showing SHCFpositive and NSS-negative ELISA were selected and subcloned at least twice. The potentially useful hybridomas were propagated in pristane-primed mice for ascites fluid production, and MAb were partially the characterized by examining
precipitation arcs formed between hybridoma ascites fluid and SHCF in IEP. Enzyme-linked immunosorbent assay (ELISA) was similar to that used in Liu c)t nl. (1992af. To screen hybridoma cultures, SHCF and NSS at 10 ,q ml-’ (total protein) and undiluted MAb supernatants were added. This was followed by urease-labelled sheep anti-mouse Ig or urease-labelled sheep antimouse IgG (CSL, Australia) in the later experiments. To examine the antibody classes; SHCF or Ag5 at 10 t_lg ml-’ and sera from SHCF- or Ag5-immunized mice were added, which was followed by rabbit antimouse IgG,, IgG>,, IgG,,, IgG,, IgA and IgM (Miles), and then urease-labelled sheep anti-rabbit IgG (CSL, Australia). The assay was developed using urease substrate and read at an absorbance of either 450 or 590 nm. Immunoelectrophoresis (IEP) of MAb ascites and mouse antisera to SHCF and Ag5 was performed as in Liu et d. (1992b). Immunoprecipitation (IP) analysis was carried out as described in Lightowlers ff al. (1989). Immunization with SHCF resulted in the production
Research Note of all major classes and subclasses of anti-hydatid antibodies in mice. A typical pattern of IgG,, IgG,,, IgG,,, IgG,, IgA and IgM antibodies is shown in Fig. 1 SHCF+ MxSHCF. Anti-hydatid IgG, and IgM antibodies were detected in ELISA at I/12,800 and I/ 800 serum dilutions and specific IgG2,, IgG,,, IgG, and IgA were present at l/40&1/800 dilutions from this particular mouse. Anti-AgS IgG antibodies were clearly present in sera of mice immunized with SHCF as these sera reacted strongly with the purified Ag5 in ELISA using a urease-labelled sheep anti-mouse IgG conjugate (CSL, Australia) (data not shown). As expected, sera from SHCF-immunized mice contained antibodies to all the major antigens of E. granulosus such as Ag5 and AgB when examined in IEP (data not shown). Immunoprecipitation analysis of mouse sera to SHCF indicated that these sera recognized various components of SHCF including those of Ag5 (24 and 38 kDa) and of AgB (8, 16 and 24 kDa) as determined in Lightowlers et al. (1989). Ag5 also stimulated substantial antibody responses in immunized mice (Fig. 1 Ag5 + MxAg5). Specific IgG, and IgM antibodies were detected at serum dilutions of l/3200 and l/400 from this particular mouse. Some IgG2, and IgGzh antibodies were also present. Mice immunized with Ag5 plus KLH showed a similar, but enhanced antibody response to Ag5 compared with mice immunized with Ag5 with initial Freund’s complete adjuvant only. IgG,, IgG,, and IgM antibodies were particularly noticeable, but IgGzb and IgGi antibodies were also produced (data not shown). Sera from non-immunized control mice did not have any detectable antibodies to hydatid antigens in both ELISA and IEP (data not shown), but they as well as SP2 ascites did precipitate a band of 51 kDa in immunoprecipitation which was probably the same as the band of 55 kDa reported by Shepherd and McManus (1987) which was postulated as sheep host heavy chain immunoglobulin. Sera of mice immunized with HPLC-purified Ag5 formed only an Arc 5 with SHCF (Liu et al., 1992b), and did not show any precipitating band with NSS in IEP (data not shown), although the presence of sheep serum components was confirmed in ELISA using Ag5 vs rabbit antiserum to NSS and NSS vs mouse antisera to Ag5 (data not shown). Mouse sera to Ag.5 precipitated bands in immunoprecipitation identical to those recognized by anti-Ag5 MAb (Lightowlers et al., 1989). Two hybrid cell lines were selected as a result of fusions between SP2 or NSI and spleen cells from SHCF-immunized mice. They were 31.15, which secretes IgGzh antibodies to AgB, and 39B3, which secretes IgG,, antibodies to AgB. Ascites fluid from
1015
these two MAb formed with SHCF an AgB-type arc in IEP and an AgB band pattern in immunoprecipitation (Lightowlers et al., 1989), and did not react with NSS in IEP (data not shown). Several IgG MAb to AgB that formed no immunoelectrophoretic bands were also produced. The identity of these MAb was determined in immunoprecipitation by their formation of a band pattern characteristic of anti-AgB MAb (Lightowlers et al., 1989). In addition, a large number of hybrid clones producing specific IgM antibodies were also obtained from the fusions between myeloma cells and spleen cells from SHCF-sensitized mice. Since anti-hydatid IgM MAb tended to form morphologically uncharacteristic bands in IEP with SHCF (data not shown), they were not pursued further in the later experiments. Despite the evidence of anti-Ag5 IgG antibodies in sera of mice immunized with SHCF, specific IgG MAb to Ag5 were not derived from these fusions. On the other hand, using donor mice immunized with Ag5, one hybrid cell line (24.14) which secretes IgG,, antibodies to Ag5 was produced. A second clone (6lAl2) which secretes IgGzh antibodies was derived from a fusion using spleen cells from Ag5and KLH-immunized mice. Ascites from both clones formed a typical Arc 5 with SHCF in IEP and an band in pattern immunoprecipitation Ag5 (Lightowlers et al., 1989). None of them showed any IEP band with NSS (data not shown). Further, a number of anti-Ag5 IgG MAb that showed no band in IEP were selected. Again, their identity was determined in immunoprecipitation by their formation of a band pattern characteristic of antiAg5 MAb (Lightowlers et al., 1989). A whole spectrum of specific antibody subclasses was detected in sera of mice injected with SHCF, especially of IgG, and IgM. Although IgG antibodies to Ag5 were present in sera from these mice in ELISA, IgG MAb to Ag5 were not obtained. It was possible that MAb to AgB might have been preferentially selected using a SHCF-based ELISA for screening hybrid cells because of stronger signals resulting from the higher proportion of AgB to Ag5 (10 to 1) in SHCF (Musiani, Piantelli, Lauriola. Arru & Pozzouli, 1978). Alternatively, it might be due to the possibility that lower numbers of anti-Ag5 B cells were induced in mice immunized with SHCF compared with those of anti-AgB B cells. It appeared that in order to facilitate the production of IgG MAb to Ag5, it might be necessary to employ a purified Ag5 to either immunize spleen donor mice or screen hybrid supernatants from fusions using spleen cells from SHCF-sensitized mice. Ag5 purified by HPLC was highly immunogenic in that it stimulated production of several different
1016
D. LIU, M. D. RKKARD and M. W. LIGHTOWLERS
antibody classes in sera of immunized mice. KLH enhanced the antibody response in AgS-sensitized mice. Sera from mice immunized with Ag5 formed only one Arc 5 band with SHCF and did not react with NSS in IEP, although mouse sera to Ag5 reacted with NSS in the more sensitive ELISA. It seemed that mice displayed a preferential immune response to Ag5 over other minor elements of hydatid antigens and sheep serum components present in HPLC-purified Ag5 preparation as assessed in IEP (Liu et al., 1992b). As a result of fusions between myelomas and spleen cells from AgS-sensitized mice, at least two IgG MAb to Ag5 were generated even though the same screening system was used. It is also of interest to observe that ascites from almost all IgM secreting hybrid cells but only some IgG secreting cells readily formed precipitating bands in IEP. Perhaps this reflects the fact that the nature of IgM molecules favours the formation of precipitation bands in comparison with that of IgG molecules. Although some of the IgG MAb failed to form any precipitation bands with SHCF in IEP, they did show similar band patterns to those formed by anti-Ag5 and AgB MAb in immunoprecipitation (Lightowlers et al., 1989). This suggests that for confirmation
of MAb
to
the
major
antigens
of E.
be a more sensitive IEP. assay than However, the demonstration of classical Arc 5 and AgB bands in IEP is an ultimate criterion for selection of MAb to Ag5 and AgB.
granulosus,
immunoprecipitation
might
Acknowledgements-This study was supported from the University of Melbourne Veterinary Fund and the National Health and Medical Council of Australia.
by grants Research Research
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