HYBRIDOMA Volume 10, Number 5, 1991 Mary Ann Liebert, Inc., Publishers
An ELISA-Method Using Magnetic Beads as Solid Phase for Rapid Quantitation of Mouse and Human Immunoglobulins 0YSTEIN
W.
R0NNING
and ANNE C. CHRISTOPHERSEN
Center for Industrial Research, P.B. 124-Blindern, 0314 Oslo 3,
Norway
ABSTRACT ELISA-method using superparamagnetic polystyrene beads solid phase, that may be used to measure the concentration of immunoglobulins in various fluids. The immunoglobulin was captured between beads coated with either rabbit, goat or sheep antibodies and the same antibody conjugated to horseradish peroxidase. The method has been used to quantitate mouse and human immunoglobulins in cell culture supernatants. Due to the rapid binding kinetcs between the beads and the soluble anigen (immunoglobulin), each incubation takes only ten minutes, allowing the whole assay to be performed within one hour.
This work describes
(Dynabeads M-450)
an
as
INTRODUCTION
Quantitation of immunoglobulins in various fluids is necessary in scientific, industrial
and clinical applications. Examples of such quantifications include determination of concentration of immunoglobulins in ascites fluid and cell culture supernatants, after purification of antibodies, leakage of ligand from immunoadsorbent columns and other solid phases, detection of contamination of immunoglobulins, detection of immunoglobulins in blood samples, etc. The most commonly used methods are radial immunodiffusion (RID) (1), spectrophotometric measurements after purification, or various radio immunoassays (RIA) or enzyme-linked immunoassays (ELISA). Each of these methods has advantages and disadvantages. RID is simple and accurate, but requires overnight incubation. Spectrophotometric measurements after purification depends on the purity and recovery obtained. RIA and ELISA take several hours to perform and are accurate if precaution are taken to control unspecific reactions. Fleming & Pen (2) has described a capture-ELISA method where mouse IgG was captured between goat anti-mouse antibodies. We have modified the captureELISA method using superparamagnetic polystyrene particles (3) as solid phase, and this method has been applied to quantitate mouse and human monoclonal antibodies in cell culture supernatants produced in this laboratory.
641
MATERIALS AND METHODS
Superparamagnetic polystyrene particles, Dynabeads M-450 (Dynal, Norway) were either used precoated with sheep anti-mouse IgG, by the manufacturer, or coated in this laboratory with rabbit anti-mouse or anti-human immunoglobulins, or goat antimouse IgM (Dako, Denmark). The coating was performed according to Dynal's protocol. Briefly, the antibody was diluted to 150 fig/ ml in 0.5 M Na-borate, pH 9.5, mixed with an equal volume of Dynabeads M-450 (tosyl activated) and rotated overnight at room temperature (20-22°C). The beads were washed five times with phosphate buffered saline (PBS), 0.015 M, pH 7.4, 0.1% BSA, the last time overnight at 4°C. After the final wash, the beads were resuspended in this buffer, to the same volume as initially mixed with the antibody solution. This gives a final concentration of 30 mg beads per ml, corresponding to 5 ug antibody per mg beads. Dynabeads M-450 (20 ul) coated with the appropriate antibody were transferred to polystyrene tubes (Nunc, Denmark), mixed with 50 ul of sample (immunoglobulins (Sigma, USA) diluted in PBS, pH 7.4, 0.05% Tween 20), and incubated on the bench for 10 min with manual shaking twice during the incubation period. The tubes were then transferred to a rack with magnets (Corning, USA) and the fluid discarded by turning the rack upside down (the beads were retained due to the magnets). The beads were washed twice with 1.0 ml PBS, pH 7.4, 0,05% Tween 20. Peroxidase conjugated sheep anti-mouse IgG (Sigma, USA), rabbit anti-mouse, anti-human Ig or goat anti-mouse IgM (Dako, Denmark) depending on the antibody coated to the beads, was diluted 1:1000 in PBS, pH 7.4, 0.05% Tween 20, added to the tubes (200 ul) and incubated 10 min as described above. The beads were washed twice with 1.0 ml PBS, pH 7.4, 0.05% Tween 20 and once with 1.0 ml Na-citrate (0.1 M pH 5.0). 200 ul of substrate (o-phenylenediamine hydrochloride (Sigma, USA), 10mg/ml in Na- citrate (0.1 M, pH 5.0), 0.015% H2O2) was added to the tubes.
After less than 5 min of incubation
on
the bench, the color
development was stopped by addition of 200 ul 4.5 M H2SO4. The tubes were put on the magnet rack and 200 ul of the solution, lacking the beads, were transferred to microtiter plates and the optical density was read at 490 nm (Immuno Reader NJ-2000). RESULTS AND DISCUSSION for applying magnetic beads as a solid phase in the capture-ELISA reduce incubation time considerably. Figure 1 shows the amount of mouse IgG bound to the beads coated with sheep anti-mouse IgG as a function of incubation time. Maximum binding was obtained after less than ten minutes of incubation. This is in line with the findings of Millan et al (4) who observed maximum binding of antigen to beads coated with monoclonal antibodies within ten minutes. However, other studies (5,6), showed the need for a longer incubation time (30 min). This is probably strongly dependent on the property of the beads; for example, shape of surface, porosity etc., and thus the accessibility of antigen to reach the attached antibodies. The above mentioned studies made use of beads other than Dynabeads M-450 used in the present work. In an attempt to use smaller and lighter beads, Dynabeads M-285 were used as solid phase. However, because of dramatically greater variability within the assay, these beads were not useful (data not shown). Besides size, the M-285 beads are more porous than the M-450 beads (Dynal A.S., personal communication) possibly resulting in problems with removal of unbound or unspecific bound material. Thus, we concluded that the Dynabeads M-450 were very promising for our purpose, and all the remaining work was done with these beads. The main
method,
reason
was to
642
1,5
E
1-°
c o a»
g
0,5
"~0
10
20
40
30
Incubation time
50
60
70
(min)
FIGURE 1 at nm as a 490 function of incubation time with mouse IgG. 20 (il Dynabeads (OD) Optical density M-450 coated with sheep anti-mouse IgG was incubated together with mouse IgG (10 ug/ml) at room temperature (20°C) with manual shaking every five minutes. At the times indicated, the beads were washed twice in PBS, 0,05% Tween 20, and stored in this buffer until all samples were
collected. AU samples were treated simultanously with peroxidase conjugated sheep anti-mouse IgG and substrate as described in Materials and Methods. Each point represents the mean of duplicate samples which did not deviate by more than 10%.
Figure 2a shows optical density (OD) at 490 nm as a function of mouse immunoglobulin concentration. There was a linear relationship (in the semilogaritmic diagram) between OD and the immunoglobulin concentration in a range of one decade. The best sensitivity for quantitating mouse IgG (~ 0.3 ug/ml) was obtained with rabbit antibody; the sheep antibody being about 5 times less sensitive. The sensitivity for detecting mouse IgM was similar to that of detecting IgG with the rabbit antibody. One explanation of the difference in sensitivity of detecting mouse IgG may be that the rabbit antibody was freshly coupled to the beads, while the beads coated with sheep antibody were made by the manufacturer and used about 6 months after the date of manufacturing (shelf-life 12 months). Some antibody may detach from the beads, and compete with the particle-bound, and therefore reduce the amount of antigen bound to the beads. The effect of this may be eliminated by washing the beads prior to use. The affinities of the two antibodies may also be different and may explain the observed difference in sensitivity. Thus, a careful selection of capturing antibody is needed if high sensitivity is to be obtained. Figure 2b shows similar curves for detection of human IgG and IgM. Also in these
cases
sensitivity sensitive.
the assay could be used in a concentration range of one decade. The best obtained for human IgG while for IgM it was about 10 times less
was
In
conclusion, the described capture-ELISA based on superparamagnetic beads (Dynabeads M-450) is very useful for quantitating immunoblobulins when reliable results are required in a relatively short time. The total time of the assay was about one hour, and it is routinely used in this laboratory for measuring the concentration of
monoclonal antibodies in cell culture supernatants.
643
,1
1
10
,01
1
,1
Immunoglobulin concentration (|ig/ml) FIGURE 2
panel). Optical density (OD) at 490 nm as a function of concentration of mouse immunoglobulins. The beads were coated with: ( ) goat anti-mouse IgM, ( ) rabbit anti-mouse IgG and ( ) sheep anti-mouse IgG. The samples lacking mouse Ig adsorbed less than 0.1 OD units, b (right panel). Same as 2a, except that the immunoglobulins were human. The beads were coated with (•) rabbit anti-human IgG or (O) rabbit anti-human IgM. The samples lacking human Ig a
(left
adsorbed less than 0.1 OD units. Each point represents the mean of duplicate samples which did not deviate by more than 10%, and the lines show a logaritmic function fitted to the points representing the straight part of the curves.
ACKNOWLEDGMENTS
The skillful technical assistance of Gro S.
acknowledged.
Haugen
and Mona Schartum is
greatly
This work was supported by grants from the Royal Norwegian Council for Scientific and Industrial Research (NTNF) and the Nordic Industrial foundation. Dynabeads M-285 was a generous gift from Dynal A.S.
REFERENCES
Mancini, G., Carbonara, A. O. and Heremans, J.F. (1965) Immunochemical
quantitation of antigens by single radial
immunodiffusion.
235.
Immunochemistry 2,
2.
Fleming, J. O. and Pen, L.B. (1988) Measurement of the concentration of murine IgG monoclonal antibody in hybridoma supernatants and ascites in absolute units by a sensitive and reliable enzyme-linked assay (ELISA). J. Immunol. Methods 110, 11.
3.
Ugelstad, J., Mfutakamba, H, Merk, P.C., Ellingsen, T., Berge, A., Schmid, R, Holm, L., Jorgedal, A., Hansen, F.K. and Nustad, K. (1985) Preparation and application of monodisperse polymer particles. J. Polym. Sei. 72, 225.
644
4.
K. and Norgaard-Pedersen, B. (1985) Highly sensitive solidassay for placental and placental-like alkaline immunoenzymometric phase phosphatases with monoclonal antibody and monodisperse polymer particles. Clin.
Millan, J.L., Nustad, Chem 31, 54.
5.
Johansen, L., Nustad, K., 0rstavik, T.B., Ugelstad, J., Bergen,
A. and
Ellingsen, T.
(1983) Excess antibody immunoassay for rat glandular kallikren. Momosized polymer particles as the preferred solid phase material. J. Immunol. Methods 59,
255. 6.
Nustad, K., Johansen, L., Ugelstad, J., Ellingsen, T. and Berge, A. (1984) Hydrophilic
monodisperse particles as solid-phase material core particles with compace particles.
shell-and
in immunoassays: Comparison! of Eur. Surg. Res 16 suppl 2, 80.
Address
reprint requests to: 0ystein Renning, Ph. D. W.
Center for Industrial Research P. b. 124-Blindern N-0314 Oslo3
Norway
645
An ELISA-Method Using Magnetic Beads as Solid Phase for Rapid Quantitation of Mouse and Human Immunoglobulins 0YSTEIN
W.
R0NNING
and ANNE C. CHRISTOPHERSEN
Center for Industrial Research, P.B. 124-Blindern, 0314 Oslo 3,
Norway
ABSTRACT ELISA-method using superparamagnetic polystyrene beads solid phase, that may be used to measure the concentration of immunoglobulins in various fluids. The immunoglobulin was captured between beads coated with either rabbit, goat or sheep antibodies and the same antibody conjugated to horseradish peroxidase. The method has been used to quantitate mouse and human immunoglobulins in cell culture supernatants. Due to the rapid binding kinetcs between the beads and the soluble anigen (immunoglobulin), each incubation takes only ten minutes, allowing the whole assay to be performed within one hour.
This work describes
(Dynabeads M-450)
an
as
INTRODUCTION
Quantitation of immunoglobulins in various fluids is necessary in scientific, industrial
and clinical applications. Examples of such quantifications include determination of concentration of immunoglobulins in ascites fluid and cell culture supernatants, after purification of antibodies, leakage of ligand from immunoadsorbent columns and other solid phases, detection of contamination of immunoglobulins, detection of immunoglobulins in blood samples, etc. The most commonly used methods are radial immunodiffusion (RID) (1), spectrophotometric measurements after purification, or various radio immunoassays (RIA) or enzyme-linked immunoassays (ELISA). Each of these methods has advantages and disadvantages. RID is simple and accurate, but requires overnight incubation. Spectrophotometric measurements after purification depends on the purity and recovery obtained. RIA and ELISA take several hours to perform and are accurate if precaution are taken to control unspecific reactions. Fleming & Pen (2) has described a capture-ELISA method where mouse IgG was captured between goat anti-mouse antibodies. We have modified the captureELISA method using superparamagnetic polystyrene particles (3) as solid phase, and this method has been applied to quantitate mouse and human monoclonal antibodies in cell culture supernatants produced in this laboratory.
641
MATERIALS AND METHODS
Superparamagnetic polystyrene particles, Dynabeads M-450 (Dynal, Norway) were either used precoated with sheep anti-mouse IgG, by the manufacturer, or coated in this laboratory with rabbit anti-mouse or anti-human immunoglobulins, or goat antimouse IgM (Dako, Denmark). The coating was performed according to Dynal's protocol. Briefly, the antibody was diluted to 150 fig/ ml in 0.5 M Na-borate, pH 9.5, mixed with an equal volume of Dynabeads M-450 (tosyl activated) and rotated overnight at room temperature (20-22°C). The beads were washed five times with phosphate buffered saline (PBS), 0.015 M, pH 7.4, 0.1% BSA, the last time overnight at 4°C. After the final wash, the beads were resuspended in this buffer, to the same volume as initially mixed with the antibody solution. This gives a final concentration of 30 mg beads per ml, corresponding to 5 ug antibody per mg beads. Dynabeads M-450 (20 ul) coated with the appropriate antibody were transferred to polystyrene tubes (Nunc, Denmark), mixed with 50 ul of sample (immunoglobulins (Sigma, USA) diluted in PBS, pH 7.4, 0.05% Tween 20), and incubated on the bench for 10 min with manual shaking twice during the incubation period. The tubes were then transferred to a rack with magnets (Corning, USA) and the fluid discarded by turning the rack upside down (the beads were retained due to the magnets). The beads were washed twice with 1.0 ml PBS, pH 7.4, 0,05% Tween 20. Peroxidase conjugated sheep anti-mouse IgG (Sigma, USA), rabbit anti-mouse, anti-human Ig or goat anti-mouse IgM (Dako, Denmark) depending on the antibody coated to the beads, was diluted 1:1000 in PBS, pH 7.4, 0.05% Tween 20, added to the tubes (200 ul) and incubated 10 min as described above. The beads were washed twice with 1.0 ml PBS, pH 7.4, 0.05% Tween 20 and once with 1.0 ml Na-citrate (0.1 M pH 5.0). 200 ul of substrate (o-phenylenediamine hydrochloride (Sigma, USA), 10mg/ml in Na- citrate (0.1 M, pH 5.0), 0.015% H2O2) was added to the tubes.
After less than 5 min of incubation
on
the bench, the color
development was stopped by addition of 200 ul 4.5 M H2SO4. The tubes were put on the magnet rack and 200 ul of the solution, lacking the beads, were transferred to microtiter plates and the optical density was read at 490 nm (Immuno Reader NJ-2000). RESULTS AND DISCUSSION for applying magnetic beads as a solid phase in the capture-ELISA reduce incubation time considerably. Figure 1 shows the amount of mouse IgG bound to the beads coated with sheep anti-mouse IgG as a function of incubation time. Maximum binding was obtained after less than ten minutes of incubation. This is in line with the findings of Millan et al (4) who observed maximum binding of antigen to beads coated with monoclonal antibodies within ten minutes. However, other studies (5,6), showed the need for a longer incubation time (30 min). This is probably strongly dependent on the property of the beads; for example, shape of surface, porosity etc., and thus the accessibility of antigen to reach the attached antibodies. The above mentioned studies made use of beads other than Dynabeads M-450 used in the present work. In an attempt to use smaller and lighter beads, Dynabeads M-285 were used as solid phase. However, because of dramatically greater variability within the assay, these beads were not useful (data not shown). Besides size, the M-285 beads are more porous than the M-450 beads (Dynal A.S., personal communication) possibly resulting in problems with removal of unbound or unspecific bound material. Thus, we concluded that the Dynabeads M-450 were very promising for our purpose, and all the remaining work was done with these beads. The main
method,
reason
was to
642
1,5
E
1-°
c o a»
g
0,5
"~0
10
20
40
30
Incubation time
50
60
70
(min)
FIGURE 1 at nm as a 490 function of incubation time with mouse IgG. 20 (il Dynabeads (OD) Optical density M-450 coated with sheep anti-mouse IgG was incubated together with mouse IgG (10 ug/ml) at room temperature (20°C) with manual shaking every five minutes. At the times indicated, the beads were washed twice in PBS, 0,05% Tween 20, and stored in this buffer until all samples were
collected. AU samples were treated simultanously with peroxidase conjugated sheep anti-mouse IgG and substrate as described in Materials and Methods. Each point represents the mean of duplicate samples which did not deviate by more than 10%.
Figure 2a shows optical density (OD) at 490 nm as a function of mouse immunoglobulin concentration. There was a linear relationship (in the semilogaritmic diagram) between OD and the immunoglobulin concentration in a range of one decade. The best sensitivity for quantitating mouse IgG (~ 0.3 ug/ml) was obtained with rabbit antibody; the sheep antibody being about 5 times less sensitive. The sensitivity for detecting mouse IgM was similar to that of detecting IgG with the rabbit antibody. One explanation of the difference in sensitivity of detecting mouse IgG may be that the rabbit antibody was freshly coupled to the beads, while the beads coated with sheep antibody were made by the manufacturer and used about 6 months after the date of manufacturing (shelf-life 12 months). Some antibody may detach from the beads, and compete with the particle-bound, and therefore reduce the amount of antigen bound to the beads. The effect of this may be eliminated by washing the beads prior to use. The affinities of the two antibodies may also be different and may explain the observed difference in sensitivity. Thus, a careful selection of capturing antibody is needed if high sensitivity is to be obtained. Figure 2b shows similar curves for detection of human IgG and IgM. Also in these
cases
sensitivity sensitive.
the assay could be used in a concentration range of one decade. The best obtained for human IgG while for IgM it was about 10 times less
was
In
conclusion, the described capture-ELISA based on superparamagnetic beads (Dynabeads M-450) is very useful for quantitating immunoblobulins when reliable results are required in a relatively short time. The total time of the assay was about one hour, and it is routinely used in this laboratory for measuring the concentration of
monoclonal antibodies in cell culture supernatants.
643
,1
1
10
,01
1
,1
Immunoglobulin concentration (|ig/ml) FIGURE 2
panel). Optical density (OD) at 490 nm as a function of concentration of mouse immunoglobulins. The beads were coated with: ( ) goat anti-mouse IgM, ( ) rabbit anti-mouse IgG and ( ) sheep anti-mouse IgG. The samples lacking mouse Ig adsorbed less than 0.1 OD units, b (right panel). Same as 2a, except that the immunoglobulins were human. The beads were coated with (•) rabbit anti-human IgG or (O) rabbit anti-human IgM. The samples lacking human Ig a
(left
adsorbed less than 0.1 OD units. Each point represents the mean of duplicate samples which did not deviate by more than 10%, and the lines show a logaritmic function fitted to the points representing the straight part of the curves.
ACKNOWLEDGMENTS
The skillful technical assistance of Gro S.
acknowledged.
Haugen
and Mona Schartum is
greatly
This work was supported by grants from the Royal Norwegian Council for Scientific and Industrial Research (NTNF) and the Nordic Industrial foundation. Dynabeads M-285 was a generous gift from Dynal A.S.
REFERENCES
Mancini, G., Carbonara, A. O. and Heremans, J.F. (1965) Immunochemical
quantitation of antigens by single radial
immunodiffusion.
235.
Immunochemistry 2,
2.
Fleming, J. O. and Pen, L.B. (1988) Measurement of the concentration of murine IgG monoclonal antibody in hybridoma supernatants and ascites in absolute units by a sensitive and reliable enzyme-linked assay (ELISA). J. Immunol. Methods 110, 11.
3.
Ugelstad, J., Mfutakamba, H, Merk, P.C., Ellingsen, T., Berge, A., Schmid, R, Holm, L., Jorgedal, A., Hansen, F.K. and Nustad, K. (1985) Preparation and application of monodisperse polymer particles. J. Polym. Sei. 72, 225.
644
4.
K. and Norgaard-Pedersen, B. (1985) Highly sensitive solidassay for placental and placental-like alkaline immunoenzymometric phase phosphatases with monoclonal antibody and monodisperse polymer particles. Clin.
Millan, J.L., Nustad, Chem 31, 54.
5.
Johansen, L., Nustad, K., 0rstavik, T.B., Ugelstad, J., Bergen,
A. and
Ellingsen, T.
(1983) Excess antibody immunoassay for rat glandular kallikren. Momosized polymer particles as the preferred solid phase material. J. Immunol. Methods 59,
255. 6.
Nustad, K., Johansen, L., Ugelstad, J., Ellingsen, T. and Berge, A. (1984) Hydrophilic
monodisperse particles as solid-phase material core particles with compace particles.
shell-and
in immunoassays: Comparison! of Eur. Surg. Res 16 suppl 2, 80.
Address
reprint requests to: 0ystein Renning, Ph. D. W.
Center for Industrial Research P. b. 124-Blindern N-0314 Oslo3
Norway
645
