HYBRIDOMA Volume 9, Number 3, 1990 Mary Ann Liebert, Inc., Publishers
A MicroELISA Useful for Determination of
Protein
A-Binding Monoclonal Antibodies
ANDERS LARSSON and RIKARD HOLMDAHL Department of Medical and Physiological Chemistry, University of Uppsala, Biomédical Centre, Uppsala, Sweden
ABSTRACT
Affinity chromatography on protein A columns is widely used for the purification of monoclonal antibodies. However, some monoclonal antibodies do not bind protein A. We have developed a sandwichELISA for determination of the protein A binding capacity of antibodies. This utilizes microtitre plates coated with protein A, and labeled chicken antibodies for detection of bound antibody. Chicken IgG is and
protein A,
background.
one can
of the few antibodies that does not react with thus be used without the problem of a high
INTRODUCTION
Protein A from Staphylococcus aureus has gained wide application as anti-IgG reagent in many immunological techniques (Forsgren et al., 1983). It reacts with most mammalian IgG and can thus be used for detection or isolation of such IgG (Lindmark et al., 1983). Affinity chromatography on protein A columns is a simple and effective method for purification of antibodies, and is now also widely used for the purifying monoclonal antibodies. However, one problem is that some monoclonal antibodies have a low affinity for protein A, such as some mouse IgG, and human IgG3 monoclonal antibodies an
(Lindmark et al., 1983). It would, therefore, be useful to be able to test the monoclonal antibodies in a rapid screening assay for their protein A binding capacity before deciding which purification technique should be used. The present assay is an ELISA which utilizes microtitre plates coated with protein A which will bind protein A-binding antibodies. Bound antibody is detected by a chicken anti-mouse IgG-alkaline phosphatase conjugate. Chicken IgG is one of the few antibodies that does not react with protein A (Langone et al., 1983), and will therefore, not cause a high background. The assay is sensitive and simple to perform and all the chemicals required are commercially available. The use of microtitre plates makes the assay well suited for screening of monoclonal antibodies. 289
MATERIALS AND METHODS
Antibodies
Affinity-purified chicken anti-mouse IgG-alkaline phosphatase conjugates (anti-mouse IgG-ALP) were provided by Immunsystem IMS AB, Uppsala, Sweden. Purified mouse IgG, (MOPC21), IgG2a (UPC10),
and IgG3 (FL0PC21) monoclonal antibodies were obChemical Company, U.S.A. The anti-mouse IgG antibody reacted with all four subclasses in immunodiffusion prior to affinity purification and conjugation.
IgG2b (M0PC141)
tained from
Sigma
Protein A Protein A
was purified from Staphylococcus aureus A676, which extracellular protein A (Lindmark et al., 1977). The bacteria was grown overnight in tryptone soy broth medium, and after centrifugation the supernatant was purified on human IgGSepharose. Purified protein A was also obtained from Pharmacia, Uppsala, Sweden.
produces
ELISA Procedure
Flat-bottom microELISA plates (Dynatech M 129 A) were coated with 200 ßl of 10 ftg/ml of protein A in 0.1 M NaHC
A MicroELISA Useful for Determination of
Protein
A-Binding Monoclonal Antibodies
ANDERS LARSSON and RIKARD HOLMDAHL Department of Medical and Physiological Chemistry, University of Uppsala, Biomédical Centre, Uppsala, Sweden
ABSTRACT
Affinity chromatography on protein A columns is widely used for the purification of monoclonal antibodies. However, some monoclonal antibodies do not bind protein A. We have developed a sandwichELISA for determination of the protein A binding capacity of antibodies. This utilizes microtitre plates coated with protein A, and labeled chicken antibodies for detection of bound antibody. Chicken IgG is and
protein A,
background.
one can
of the few antibodies that does not react with thus be used without the problem of a high
INTRODUCTION
Protein A from Staphylococcus aureus has gained wide application as anti-IgG reagent in many immunological techniques (Forsgren et al., 1983). It reacts with most mammalian IgG and can thus be used for detection or isolation of such IgG (Lindmark et al., 1983). Affinity chromatography on protein A columns is a simple and effective method for purification of antibodies, and is now also widely used for the purifying monoclonal antibodies. However, one problem is that some monoclonal antibodies have a low affinity for protein A, such as some mouse IgG, and human IgG3 monoclonal antibodies an
(Lindmark et al., 1983). It would, therefore, be useful to be able to test the monoclonal antibodies in a rapid screening assay for their protein A binding capacity before deciding which purification technique should be used. The present assay is an ELISA which utilizes microtitre plates coated with protein A which will bind protein A-binding antibodies. Bound antibody is detected by a chicken anti-mouse IgG-alkaline phosphatase conjugate. Chicken IgG is one of the few antibodies that does not react with protein A (Langone et al., 1983), and will therefore, not cause a high background. The assay is sensitive and simple to perform and all the chemicals required are commercially available. The use of microtitre plates makes the assay well suited for screening of monoclonal antibodies. 289
MATERIALS AND METHODS
Antibodies
Affinity-purified chicken anti-mouse IgG-alkaline phosphatase conjugates (anti-mouse IgG-ALP) were provided by Immunsystem IMS AB, Uppsala, Sweden. Purified mouse IgG, (MOPC21), IgG2a (UPC10),
and IgG3 (FL0PC21) monoclonal antibodies were obChemical Company, U.S.A. The anti-mouse IgG antibody reacted with all four subclasses in immunodiffusion prior to affinity purification and conjugation.
IgG2b (M0PC141)
tained from
Sigma
Protein A Protein A
was purified from Staphylococcus aureus A676, which extracellular protein A (Lindmark et al., 1977). The bacteria was grown overnight in tryptone soy broth medium, and after centrifugation the supernatant was purified on human IgGSepharose. Purified protein A was also obtained from Pharmacia, Uppsala, Sweden.
produces
ELISA Procedure
Flat-bottom microELISA plates (Dynatech M 129 A) were coated with 200 ßl of 10 ftg/ml of protein A in 0.1 M NaHC
