Journal oflmmunologicalMethods, 130 (1990) 141-147 Elsevier
141
JIM05594
Discrimination by rabbit anti-idiotypic antibodies of two murine IgM monoclonal antibodies directed against lipid A J.J. Cornelissen
2, T.A.M.
Oosterlaken 1, j. Schellekens 1, R. Torensma 1, M. Rozenberg-Arska 1, C.A. Kraaijeveld i and J. Verhoef 1
I Eijkman-l¥inkler Laboratory for Microbiology, and 2 Department of Internal Medicine, University Hospital Utrecht, The Netherlands
(Received 2 August 1989, revised received 8 January 1990, accepted 16 February 1990)
Two murine IgM monoclonal antibodies (MAs) directed against the lipid A portion of bacterial lipopolysaccharide (LPS) were compared in their binding to Re LPS and lipid A and their idiotypic make-up with rabbit anti-idiotypic sera. Horseradish peroxidase (HRPO)-labelled MAs 8-2 and 26-20 bound equally well to Re LPS. The binding of HRPO-labelled MA 8-2 to synthetic lipid A was low compared to the relatively strong binding of labelled 26-20. The MAs proved to be competitive in a competition binding assay (CBA) with Re LPS as coating antigen. Rabbit immune sera were raised against individual MAs. Anti-idiotypic antibodies (anti-id Abs) were detected with two sensitive enzyme immunoassays (EIA)" a solid-phase EIA and an inhibition EIA. The rabbit antisera proved to be idiotype specific, indicating that both MAs recognize separate epitopes. We expect that anti-id Abs will prove to be of value for the differentiation of panels of LPS specific MAs. Key words: Lipopolysaccharide; Lipid A; Monoclonal antibody; Anti-idiotypic antibody
Introduction
Antibodies directed against the lipid A-coreoligosaccharide part of lipopolysaccharides (LPS) may afford protection against gram-negative septic shock (Ziegler et al., 1982; Baumgartner et al., 1985). Therefore it is of great interest to identify the protection inducing epitopes on these lipid
Correspondence to: J.J. Cornelissen, Department of Internal Medicine and Clinical Microbiology, Rm. G.04.515, University Hospital Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. Abbreviations. LPS, lipopolysaccharide; MA, monoclonal antibody; Anti-id Abs, anti-idiotypic antibodies; CBA, competition binding assay; EIA, enzyme immunoassay; HRPO, horseradish peroxidase.
A-core-oligosaccharide structures, which are shared by a variety of gram-negative bacilli. Various authors have used monoclonal antibodies (MAs) to identify these important immunodeterminants (Kirkland et al., 1986; Bogard et al., 1987; Pollack et al., 1989) but there remains the problem of dissecting the fine determinant specificity of the different antibodies. We have produced a number of MAs directed against various epitopes of the core-glycolipid (De Jongh-Leuvenink et al., 1986; Erich et al., 1989b) and some of them are currently being tested in vivo. In this paper we describe the development of anti-idiotypic antibodies (anti-id Abs) against two IgM MAs, MA 8-2 and MA 26-20, directed against the lipid A portion of LPS. We show in this study that MA 8-2 and MA 26-20 are competitive in a competition binding assay (CBA). However, as
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
142 revealed by the use of anti-id Abs in different assays, their idiotypes proved to be different.
Materials and methods
A n tigens Re LPS of Salmonella minnesota R595 was isolated using the phenol-chloroform-petroleum ether extraction methoo as described by Galanos et al. (1977). Lipid A was prepared from E. coli strain F 515 by hydrolysis with sodium acetate (0.1 M, pH 4.4, 1 h, 100°C), according to Brade et al. (1985). Before each test, Re LPS or lipid A were freshly diluted after ultrasonic treatment (3 rain) of the stock solution. Monoclonal antibodies The production and characterization of IgM MAs 8-2 and 26-20 are described elsewhere (Erich et al., 1989b). Both MAs are produced by hybridomas of B A L B / c mouse spleen cells and the mouse myeloma cell line Sp2/0-Agl4. MA 8-2 was induced by the Re-mutant of Salmonella typhimurium and MA 26-20 was induced by synthetic lipid A. MA 8-2 and MA 26-20 were purified by gel filtration of mouse ascitic fluids using a column of Sephacryl S-300 (1 x 100 cm) Enzyme labelling of MA Horseradish peroxidase (HRPO) was conjugated to purified MA by the periodate method (Nakane et al., 1974). The conjugates were stored at - 2 0 ° C. Immediately before use the conjugates were diluted in phosphate-buffered saline (PBS) with 0.05% Tween 20 (PBST). Final dilutions ranged from 1/5000 to 1/10,000. Immunization of rabbits Purified MA, 1 mg in 1 ml PBS, was mixed with 1 ml of Freund's complete adjuvant (FCA) and intracutaneously injected at six different sites into the back of first generation off-spring of New Zealand rabbits. Booster injections (1 mg MA) were given with incomplete Freund's adjuvant (1 ml) on day 28. Serum was obtained 10 days later and stored in small aliquots at - 2 0 ° C. The immune sera were absorbed with normal mouse serum (NMS) from B A L B / c mice to remove
anti-allotypic and anti-isotype antibodies as previously described (Oosterlaken et al., 1988). Briefly: four parts of NMS were mixed with one part of rabbit serum and incubated overnight at 4°C. Absorptions of immune sera with MAs were similarly performed.
Enzyme immunoassay (EIA) of LPS with HRPOlabelled MAs To each well of a 96 well polyvinyl (PVC) microtiter plate (Flow Laboratories, the Netherlands) 100 ng of Re LPS in 0.1 ml PBS of pH 7.2 were added and incubated overnight at 4 ° C. Lipid A was coated by HCi precipitation as described previously by Erich et al. (1989a). To each well 500 ng lipid A in 0.1 ml double distilled water were added. Subsequently 25 ~tl cold HCI (0.4 N) were added and the lipid A precipitated over 30 rain at 4 ° C . The plates were centrifuged (2000 rpm, 5 min, 4° C) and then washed with cold HCl (0.1 N) for 15 min at 4 C. After centrifugation the plates were washed with water and PBST alternately. Thereafter the plates were gently shaken dry and serial dilutions of HRPO-labelled MA 8-2 or 26-20 were added (0.1 ml PBST/well). After incubating for 1 h at 37°C. the plates were washed with tap water and PBST alternately. The amount of bound peroxidase was visualized by incubating the wells with 0.1 ml. of a solution of 3,3',5,5'-tetramethylbenzidine (Sigma Chemical Co.) and H202. After a 30 min incubation at room temperature, the enzyme reaction was stopped with 0.1 ml of 0.18 M H2SO4/well and the peroxidase was qiaantified by measuring the optical density at 450 nm with a titertek Multiscan (Flow Laboratories, Irvine, Scotland, U.K.) Competition binding assay (CBA) Competition among MAs for determinants on Re LPS was assayed by an enzyme immunoassay (EIA). To each well of a 96 well plate (Flow Laboratories, The Netherlands) 100 ng of Re LPS in 0.1 ml PBS of pH 7.2 were added and incubated overnight at 4 ° C. After coating, the plates were gently shaken dry, graded doses of purified MAs in 0.1 ml PBST were added and the plates incubated for 1 h at 37°C. Subsequently, HRPOlabelled MAs, diluted to suitable concentrations,
143
were added in 0.1 ml portions per well and incubated for 30 min at 37 ° C. The plates were then washed with tapwater and PBST alternatively. The amount of bound peroxidase was determined as described above.
Solid-phase EIA for the detection of anti-idiotypic antibodies Plates (catalogue no. 3596, Costar Plastics, Cambridge, MA, U.S.A.) were coated overnight at 4 ° C with 0.2 /xg of purified MA in 0.1 ml carbonate buffer of p H 9.6. After coating, the buffer fluid was discarded and 0.1 ml PBS + 0.5% gelatine supplemented with 3.5% bovine serum albumin (BSA), prewarmed to 37 ° C, was added to each well and the plate incubated for 30 rain. Next the plates were washed with PBST and tapwater alternately. Subsequently 0.1 ml aliquots of absorbed rabbit anti-idiotypic serum, diluted in PBST, were added to the wells and the plates incubated for 1 h at 3 7 ° C followed by careful rinsing of the wells with tap water. Thereafter 0.1 ml portions of HRPO-labelled MA (diluted 1/10,000 in PBST) were added and the plates incubated for 1 h at 37 o C. The amount of bound peroxidase was determined as described for the competition ELISA. Inhibition EIA Plates (Flow Laboratories) were coated with Re LPS (100 /zl/well of 1 /xg/ml solution in PBS). Rabbit antisera were used as either nonabsorbed or absorbed reagents. Dilutions of rabbit anti-idiotypic serum, diluted in PBST were mixed with suitable concentrations of HRPO-labelled MAs and incubated for 1 h at 37 ° C in separate 96-well (non-blocked) plates. Subsequently 0.1 ml aliquots of these rabbit anti-sera mixed with H R P O labelled MAs were pipetted into plates coated with Re LPS followed by a 1 h incubation at 37°C. Afterwards the plates were washed and developed as described above.
(B)
12
\\ 04
\xxx m
I 3
I 4
i 5
-1°log dilution
3
4
of labelled
5 rna
Fig. 1. Binding of MA 8-2 and MA 26-20 to Re LPS and lipid A. Plates coated with Re LPS (0.1 p.g/ml, A) or lipid A (0.5 ~tg/ml, B) were incubated with serial dilutions of HRPOlabelled MA 8-2 ( o ) or HRPO-labelled MA 26-20 (zx). Mean (n = 2) absorbance is given at each dilution of labelled MA.
were detected by EIA using serial dilutions (1/1000, 1/10,000 and 1/100,000) of HRPO-coupled MA 8-2 or 26-20. The results are presented in Fig. 1. Both MAs were bound equally well to Re 100
(A)
8o
40
~
.-g
o
~.
loo
°u
80
E
~"
2o I
o
I
I
1
i
(") "-,
40O
4O
dose of unlabelled
4
04
O.O4
ma per well ~ng)
Fig. 2. Competition between MA 8-2 and MA 26-20 for determinants on Re LPS. Plates coated with Re LPS (0.1 ~tg/ml) were incubated with either purified MA 8-2 (
141
JIM05594
Discrimination by rabbit anti-idiotypic antibodies of two murine IgM monoclonal antibodies directed against lipid A J.J. Cornelissen
2, T.A.M.
Oosterlaken 1, j. Schellekens 1, R. Torensma 1, M. Rozenberg-Arska 1, C.A. Kraaijeveld i and J. Verhoef 1
I Eijkman-l¥inkler Laboratory for Microbiology, and 2 Department of Internal Medicine, University Hospital Utrecht, The Netherlands
(Received 2 August 1989, revised received 8 January 1990, accepted 16 February 1990)
Two murine IgM monoclonal antibodies (MAs) directed against the lipid A portion of bacterial lipopolysaccharide (LPS) were compared in their binding to Re LPS and lipid A and their idiotypic make-up with rabbit anti-idiotypic sera. Horseradish peroxidase (HRPO)-labelled MAs 8-2 and 26-20 bound equally well to Re LPS. The binding of HRPO-labelled MA 8-2 to synthetic lipid A was low compared to the relatively strong binding of labelled 26-20. The MAs proved to be competitive in a competition binding assay (CBA) with Re LPS as coating antigen. Rabbit immune sera were raised against individual MAs. Anti-idiotypic antibodies (anti-id Abs) were detected with two sensitive enzyme immunoassays (EIA)" a solid-phase EIA and an inhibition EIA. The rabbit antisera proved to be idiotype specific, indicating that both MAs recognize separate epitopes. We expect that anti-id Abs will prove to be of value for the differentiation of panels of LPS specific MAs. Key words: Lipopolysaccharide; Lipid A; Monoclonal antibody; Anti-idiotypic antibody
Introduction
Antibodies directed against the lipid A-coreoligosaccharide part of lipopolysaccharides (LPS) may afford protection against gram-negative septic shock (Ziegler et al., 1982; Baumgartner et al., 1985). Therefore it is of great interest to identify the protection inducing epitopes on these lipid
Correspondence to: J.J. Cornelissen, Department of Internal Medicine and Clinical Microbiology, Rm. G.04.515, University Hospital Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. Abbreviations. LPS, lipopolysaccharide; MA, monoclonal antibody; Anti-id Abs, anti-idiotypic antibodies; CBA, competition binding assay; EIA, enzyme immunoassay; HRPO, horseradish peroxidase.
A-core-oligosaccharide structures, which are shared by a variety of gram-negative bacilli. Various authors have used monoclonal antibodies (MAs) to identify these important immunodeterminants (Kirkland et al., 1986; Bogard et al., 1987; Pollack et al., 1989) but there remains the problem of dissecting the fine determinant specificity of the different antibodies. We have produced a number of MAs directed against various epitopes of the core-glycolipid (De Jongh-Leuvenink et al., 1986; Erich et al., 1989b) and some of them are currently being tested in vivo. In this paper we describe the development of anti-idiotypic antibodies (anti-id Abs) against two IgM MAs, MA 8-2 and MA 26-20, directed against the lipid A portion of LPS. We show in this study that MA 8-2 and MA 26-20 are competitive in a competition binding assay (CBA). However, as
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
142 revealed by the use of anti-id Abs in different assays, their idiotypes proved to be different.
Materials and methods
A n tigens Re LPS of Salmonella minnesota R595 was isolated using the phenol-chloroform-petroleum ether extraction methoo as described by Galanos et al. (1977). Lipid A was prepared from E. coli strain F 515 by hydrolysis with sodium acetate (0.1 M, pH 4.4, 1 h, 100°C), according to Brade et al. (1985). Before each test, Re LPS or lipid A were freshly diluted after ultrasonic treatment (3 rain) of the stock solution. Monoclonal antibodies The production and characterization of IgM MAs 8-2 and 26-20 are described elsewhere (Erich et al., 1989b). Both MAs are produced by hybridomas of B A L B / c mouse spleen cells and the mouse myeloma cell line Sp2/0-Agl4. MA 8-2 was induced by the Re-mutant of Salmonella typhimurium and MA 26-20 was induced by synthetic lipid A. MA 8-2 and MA 26-20 were purified by gel filtration of mouse ascitic fluids using a column of Sephacryl S-300 (1 x 100 cm) Enzyme labelling of MA Horseradish peroxidase (HRPO) was conjugated to purified MA by the periodate method (Nakane et al., 1974). The conjugates were stored at - 2 0 ° C. Immediately before use the conjugates were diluted in phosphate-buffered saline (PBS) with 0.05% Tween 20 (PBST). Final dilutions ranged from 1/5000 to 1/10,000. Immunization of rabbits Purified MA, 1 mg in 1 ml PBS, was mixed with 1 ml of Freund's complete adjuvant (FCA) and intracutaneously injected at six different sites into the back of first generation off-spring of New Zealand rabbits. Booster injections (1 mg MA) were given with incomplete Freund's adjuvant (1 ml) on day 28. Serum was obtained 10 days later and stored in small aliquots at - 2 0 ° C. The immune sera were absorbed with normal mouse serum (NMS) from B A L B / c mice to remove
anti-allotypic and anti-isotype antibodies as previously described (Oosterlaken et al., 1988). Briefly: four parts of NMS were mixed with one part of rabbit serum and incubated overnight at 4°C. Absorptions of immune sera with MAs were similarly performed.
Enzyme immunoassay (EIA) of LPS with HRPOlabelled MAs To each well of a 96 well polyvinyl (PVC) microtiter plate (Flow Laboratories, the Netherlands) 100 ng of Re LPS in 0.1 ml PBS of pH 7.2 were added and incubated overnight at 4 ° C. Lipid A was coated by HCi precipitation as described previously by Erich et al. (1989a). To each well 500 ng lipid A in 0.1 ml double distilled water were added. Subsequently 25 ~tl cold HCI (0.4 N) were added and the lipid A precipitated over 30 rain at 4 ° C . The plates were centrifuged (2000 rpm, 5 min, 4° C) and then washed with cold HCl (0.1 N) for 15 min at 4 C. After centrifugation the plates were washed with water and PBST alternately. Thereafter the plates were gently shaken dry and serial dilutions of HRPO-labelled MA 8-2 or 26-20 were added (0.1 ml PBST/well). After incubating for 1 h at 37°C. the plates were washed with tap water and PBST alternately. The amount of bound peroxidase was visualized by incubating the wells with 0.1 ml. of a solution of 3,3',5,5'-tetramethylbenzidine (Sigma Chemical Co.) and H202. After a 30 min incubation at room temperature, the enzyme reaction was stopped with 0.1 ml of 0.18 M H2SO4/well and the peroxidase was qiaantified by measuring the optical density at 450 nm with a titertek Multiscan (Flow Laboratories, Irvine, Scotland, U.K.) Competition binding assay (CBA) Competition among MAs for determinants on Re LPS was assayed by an enzyme immunoassay (EIA). To each well of a 96 well plate (Flow Laboratories, The Netherlands) 100 ng of Re LPS in 0.1 ml PBS of pH 7.2 were added and incubated overnight at 4 ° C. After coating, the plates were gently shaken dry, graded doses of purified MAs in 0.1 ml PBST were added and the plates incubated for 1 h at 37°C. Subsequently, HRPOlabelled MAs, diluted to suitable concentrations,
143
were added in 0.1 ml portions per well and incubated for 30 min at 37 ° C. The plates were then washed with tapwater and PBST alternatively. The amount of bound peroxidase was determined as described above.
Solid-phase EIA for the detection of anti-idiotypic antibodies Plates (catalogue no. 3596, Costar Plastics, Cambridge, MA, U.S.A.) were coated overnight at 4 ° C with 0.2 /xg of purified MA in 0.1 ml carbonate buffer of p H 9.6. After coating, the buffer fluid was discarded and 0.1 ml PBS + 0.5% gelatine supplemented with 3.5% bovine serum albumin (BSA), prewarmed to 37 ° C, was added to each well and the plate incubated for 30 rain. Next the plates were washed with PBST and tapwater alternately. Subsequently 0.1 ml aliquots of absorbed rabbit anti-idiotypic serum, diluted in PBST, were added to the wells and the plates incubated for 1 h at 3 7 ° C followed by careful rinsing of the wells with tap water. Thereafter 0.1 ml portions of HRPO-labelled MA (diluted 1/10,000 in PBST) were added and the plates incubated for 1 h at 37 o C. The amount of bound peroxidase was determined as described for the competition ELISA. Inhibition EIA Plates (Flow Laboratories) were coated with Re LPS (100 /zl/well of 1 /xg/ml solution in PBS). Rabbit antisera were used as either nonabsorbed or absorbed reagents. Dilutions of rabbit anti-idiotypic serum, diluted in PBST were mixed with suitable concentrations of HRPO-labelled MAs and incubated for 1 h at 37 ° C in separate 96-well (non-blocked) plates. Subsequently 0.1 ml aliquots of these rabbit anti-sera mixed with H R P O labelled MAs were pipetted into plates coated with Re LPS followed by a 1 h incubation at 37°C. Afterwards the plates were washed and developed as described above.
(B)
12
\\ 04
\xxx m
I 3
I 4
i 5
-1°log dilution
3
4
of labelled
5 rna
Fig. 1. Binding of MA 8-2 and MA 26-20 to Re LPS and lipid A. Plates coated with Re LPS (0.1 p.g/ml, A) or lipid A (0.5 ~tg/ml, B) were incubated with serial dilutions of HRPOlabelled MA 8-2 ( o ) or HRPO-labelled MA 26-20 (zx). Mean (n = 2) absorbance is given at each dilution of labelled MA.
were detected by EIA using serial dilutions (1/1000, 1/10,000 and 1/100,000) of HRPO-coupled MA 8-2 or 26-20. The results are presented in Fig. 1. Both MAs were bound equally well to Re 100
(A)
8o
40
~
.-g
o
~.
loo
°u
80
E
~"
2o I
o
I
I
1
i
(") "-,
40O
4O
dose of unlabelled
4
04
O.O4
ma per well ~ng)
Fig. 2. Competition between MA 8-2 and MA 26-20 for determinants on Re LPS. Plates coated with Re LPS (0.1 ~tg/ml) were incubated with either purified MA 8-2 (
