- Email: [email protected]
[51] Measurement of monoclonal immunoglobulin concentrations in hybridoma cultures by competitive inhibition enzyme immunoassay
[51]
ANTIBODY
MEASUREMENT
IN H Y B R I D O M A C U L T U R E S
541
The EIFA described here has several distinct advantages over other assay systems, such as the RIA. When read visually, this EIFA takes no more than 2 hr to complete. These filter plates are more sensitive than equivalent poly(vinyl chloride) plate assays, they do not require a lengthy pretreatment of the assay plate in order to reduce nonspecific adsorption of antibody, and washes can be completed in less than 1 min without centrifugation, or poly-L-lysine or glutaraldehyde fixation which may alter epitopes. Moreover, cells are not lost or reduced as in the centrifugationwash steps of other assays. Acknowledgment We would like to acknowledge the support of NIH Grants EY-0393 and CA-32047.
[51] M e a s u r e m e n t of M o n o c l o n a l I m m u n o g l o b u l i n C o n c e n t r a t i o n s in H y b r i d o m a C u l t u r e s b y C o m p e t i t i v e Inhibition E n z y m e I m m u n o a s s a y
By
KENNETH W. HUNTER, JR. a n d JOHN
M. BOSWORTH, JR.
The selection of hybridomas most often involves a determination of specificity, and less often affinity. Still less often addressed is the secretion rate of monoclonal antibodies by particular hybridoma clones. This latter issue may be of secondary but practical importance as monoclonal antibodies are further commercialized. Although mouse monoclonal antibodies can be produced in large scale by the ascites method, human monoclonal antibody production presently relies on mass in vitro culture. By selecting clones that exhibit the highest rates of antibody secretion, one can substantially improve the yield per volume of culture. This chapter describes a competitive inhibition enzyme immunoassay1 for accurately measuring the levels of immunoglobulin encountered in hybridoma cultures, the parameters that govern performance of the assay, and the advantages of the method over antigen-capture enzyme immunoassay. Materials and Methods
Reagents. Alkaline phosphatase-conjugated goat anti-rabbit IgG and p-nitrophenyl phosphate were obtained from Sigma Chemical Co. (St. t j. M. Bosworth, A. A. Brimfield, J. A. Naylor, and K. W. Hunter, J. Immunol. Methods 62, 331 (1983).
METHODS IN ENZYMOLOGY, VOL. 121
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
542
MONOCLONAL ANTIBODIES
[51]
Louis, MO). Rabbit antibodies to mouse IgGi (Lot RFC028), and the myeloma protein MOPC 21 (IgG~, Lot DG071) were obtained from Litton Bionetics (Kensington, MD). Human IgG (Lot 25), goat anti-human IgG (Lot BD1 l-B), and alkaline phosphatase-conjugated rabbit anti-goat IgG were obtained from Kirkegaard & Perry Laboratories (Rockville, MD). Competitive Inhibition Enzyme Immunoassay (CIEIA). The solidphase adsorbant used in the CIEIA was a 96-well round-bottomed polystyrene microtiter plate (Dynatech Laboratories, Alexandria, VA). It should be noted that some plastic microtiter plates work very poorly in the CIEIA. The critical importance of reagent concentrations on the performance of the CIEIA will be illustrated in the experimental section. Each well of the microtiter plate is incubated with 50/zl of an optimal concentration of the immunoglobulin (Ig) to be quantified (e.g., mouse IgGl), diluted in 0.1 M NaHCO3 buffer (pH 9.6). The time and temperature of the incubation are not critical; we routinely use overnight incubations at 4°. The fluid phase of the competitive inhibition reaction is set up in a separate microtiter plate. We recommend washing this plate with phosphate-buffered saline containing 0.5% polyoxyethylene sorbitan monolaurate (PBS-T) to reduce adsorption of reagents to the plastic. The fluid-phase reaction consists of equal volumes (50/zl) of a known standard concentration of Ig (or your sample that contains an unknown concentration) and an antibody to that Ig. Routinely, for generating the standard curve we make serial log dilutions of Ig in the same vehicle as the test sample (e.g., culture medium), beginning at 100/zg/ml. The anti-Ig is used in a concentration that does not saturate the binding sites of an Ig-coated microwell. The mixtures are incubated for 1 hr at 4°. The Ig-coated plates are washed by five cycles of filling and emptying all wells with cold PBS-T. Aliquots of 50/zl of the Ig-anti-Ig mixtures are transferred to the Ig-coated plates and incubated for 30 min at 4 °. The Ig in the fluid phase competes with the solid-phase-bound Ig for the anti-Ig. The higher the concentration of Ig in the fluid phase the less anti-Ig will bind to the solid phase. The amount of solid-phase-bound anti-Ig is detected by an enzyme-tagged second antibody. Although reasonable results can be obtained with an enzyme-labeled first antibody, we have found that the double antibody method provides an amplified signal. After washing as described above, 50 /zl of an optimal PBS-T dilution of an alkaline phosphatase-conjugated antibody to the anti-Ig is added for another 30 min at 4°. Finally, the plates are washed and 100/zl of substrate (1 mg/ml, p-nitrophenyl phosphate in 0.1 M ethanolamine buffer, pH 9.6) is added to each well. The enzymatic reaction is allowed to proceed for 30 min at room temperature, and then the colored product in each well is measured spectrophotometrically at 405 nm with a Titertek Multiscan
[51]
ANTIBODY MEASUREMENT IN HYBRIDOMA CULTURES
543
micro-ELISA reader (Flow Laboratories, Vienna, VA). The absorbance is inversely related to the concentration of Ig in the fluid phase, and a standard curve is prepared by plotting Ig concentration versus absorbance. Each point on the standard curve represents the average absorbance of six replicates. Maximum absorbance is that obtained by incubating only vehicle with anti-Ig. Linear regression analysis of these data usually yield correlation coefficients greater than 0.90. To determine the Ig concentration of a test sample, the mean absorbance value of the test sample (hybridoma culture supernatant) is entered as the y value in the regression equation of the standard curve, and the corresponding x value is calculated. This analysis system eliminates the subjectivity inherent in deriving data values from manually plotted standard curves, and also rectifies the small deviation from linearity seen in most standard curves. Antigen-Capture Enzyme Immunoassay (ACEIA). The solid-phase adsorbant and washing procedure were the same as used in the CIEIA. Plates were coated overnight at 4 ° with 50/xl of an optimal dilution of antiIg in 0.1 M NaHCO3 buffer (pH 9.6). A standard curve was generated by incubating serial log dilutions (50/xl) of Ig standard in anti-Ig-coated microtiter plates for 30 min at 4°. After washing, the "captured" Ig was sandwiched by the addition of the same anti-Ig in PBS-T for 30 min at 4 °. The remainder of the ACEIA is exactly the same as that described for the CIEIA. Optimization of the CIEIA. To illustrate the critical importance of optimization of reagent concentrations in the performance of the CIEIA, an assay of human IgG will be used. In this example, purified human IgG was adsorbed to the solid phase, and a goat anti-human IgG along with the same purified human IgG were the fluid-phase reagents. The solid-phase binding was developed with a rabbit anti-goat lgG that was tagged with alkaline phosphatase. Reaction conditions were as described in the Materials and Methods section. In this optimization experiment, microtiter plates were coated with 10, 1, or 0.1 /zg human IgG/ml. Four different concentrations of goat antihuman IgG (10, 5, 1, 0.1/xg/ml) and rabbit anti-goat IgG (10, 2, 1, 0.2/zg/ ml) were also tested. The results of this optimization experiment are shown in Fig. 1. Each reagent concentration was critical to the CIEIA. As we described in an earlier paper, j the slope of the standard curve should be as steep as possible so that variability in absorbance values plotted arithmetically on the y axis will not yield unwieldly variabilities in Ig concentrations plotted logarithmically on the x axis. It can be seen in Fig. 1 that a human IgG coating concentration of 10/zg/ml yielded the highest absolute absorbance values, but the slopes of the curves were quite fiat
544
MONOCLONAL ANTIBODIES
1.5 g --~ 1.(1
j
[51]
b:
g
,,.,~
c_
c~ , . 4 ~
b
'~ 0.5 ,,,7,
Jia:illLilll
~
II~P--4P'~r.~k-IP"~ld
E ~
F
G
aH
I
J
K
L
E 1.5 g
----1.0 o
'~ 0.~
E_1.5 g
~0.5 ,
t
1000 10 0,1 0.001 1000 10 0.1 0.001 1000 10 0.1 0.001 1000 10 0.1 0,001
[pg/ml] FIG. 1. Optimization of reagent concentrations for CIEIA of human IgG. The coating concentrations of human IgG were 10/.tg/ml (panels A-D), 1 /~g/ml (panels E-H), and 0.1 /~g/ml (panels I-L). Goat anti-human IgG concentrations were 10/xg/ml (panels A, E, and I), 5/zg/ml (panels B, F, and J), 1 p.g/ml (panels C, G, and K), and 0.1 p~g/ml (panels D, H, and L). Rabbit anti-goat IgG concentrations were 10/xg/ml (a), 5/zg/ml (b), 2/xg/ml (c), and 0.2 tzg/ml (d). Each point is the mean of six replicates.
[51]
ANTIBODY MEASUREMENT IN HYBRIDOMA CULTURES
545
regardless of the concentrations of the other reagents. When plates were coated with 0.1/zg/ml, the absolute absorbance values were substantially lower, and no combination of reagents produced a reasonable standard curve. At what would be considered the optimal human IgG coating concentration (1 /xg/ml), standard curves with the desired characteristics were generated. However, as can easily be seen the concentrations of goat anti-human IgG (panels E - H ) and rabbit anti-goat IgG (curves a-d on each panel) played a critical role. It appeared that with these particular reagents, 1 or 5/xg of goat anti-human IgG per milliliter coupled only with the highest concentration of rabbit anti-goat IgG (10/xg/ml) yielded good standard curves. These data point out the importance of optimization experiments in the development of CIEIA methods. Comparison of CIEIA and ACEIA. Another method for quantifying immunoglobulins is ACEIA, or sandwich immunoassay. We have compared this method with CIEIA and find the latter to be superior. For example, in Fig. 2 we show standard curves obtained for mouse IgGj 1.4
1.2
|
1.0
0.8 oo
0.6
0.4
0.2
I
I
100
10
I
I
I
I
I
1 0.1 0.01 0,001 0.0001 IgG1 (pg~ml)
FIG. 2. Comparison of standard curves for mouse monoclonal IgG~ concentrations by CIEIA (0) and ACEIA (O). Each point is the mean -+ one standard deviation of six replicates.
546
MONOCLONAL ANTIBODIES
[51]
under optimized conditions (for both CIEIA and ACEIA). In both assay modes, linear curves from approximately 100 /xg/ml to 10 ng/ml were obtained. Though similar in sensitivity, the curves had very different slopes; the CIEIA curve was steeper than the ACEIA curve. Since both methods had been optimized with respect to reagent concentrations, it is likely that the difference in slope is a function of the mode of assay. This difference in slope resulted in an average standard deviation from the mean of each point on the ACEIA curve of 0.06 absorbance units, compared to only 0.02 for the CIEIA. Data from several experiments indicated excellent reproducibility in standard curves, and confirmed the larger variability associated with each point on the ACEIA curve. Together, the steeper slope and lower variability of the CIEIA curve make this assay method superior to the ACEIA. As an illustration, if the variability of IgG1 concentrations at the point on Fig. 2 where the curves intersect (0.95 absorbance units and 0.9/~g/ml IgG0 is assessed, the range of one standard deviation above and below the mean is 0.02-0.33/zg/ml for ACEIA, but only 0.08-0.11/xg/ml for CIEIA. Therefore, the precision with which IgG1 concentrations can be measured is greater in the CIEIA. Discussion The capability of accurately measuring the rate of monoclonal antibody secretion by hybridoma cultures should facilitate the selection of clones with the greatest production potential. The concentrations of Ig found in hybridoma cultures produced in our laboratory range from 25/~g/ ml to I ng/ml, with an average of approximately 1 /xg/ml. Probably the most common method for quantifying Ig is by single radial immunodiffusion, 2,3 but this method cannot detect the low levels found in hybridoma supernatants. Other techniques which have been employed to detect Ig in cultures include immunoelectrophoresis, 4,5 hemagglutination inhibition, 6 and reverse plaquing, 7 all of which are semiquantitative. Radioimmunoassay for Ig is sensitive and quantitative, but because it requires the use of isotopes, 8,9 the enzyme-linked immunosorbent assay (ELISA) originally 2 G. Mancini, A. O. Carbonara, and J. F. Heremans, lmmunochemistry 2, 235 (1965). 3 j._p. Vaerman, this series, Vol. 73, Part B, p. 291. 4 j. L. Fahey, I. Finegold, A. S. Rabson, and R. A. Manaker, Science 152, 1259, 1966. 5 R. van Furth, H. R. E. Schuit, and W. Hijmans, Immunology 11, 1 (1966). 6 F. P. Siegal and M. Siegal, J. lmmunol. 118, 642 (1977). 7 G. A. Molinaro and S. Dray, Nature (London) 248, 515 (1974). 8 T. Hirano, T. Kuriani, T. Kishimoto, and Y. Yamamura, J. Immunol. 119, 1235 (1977). 9 S. M. McLachland, B. R. Smith, and R. Hall, J. Immunol. Methods 21, 212 (1970).
[51]
ANTIBODY MEASUREMENT IN HYBRIDOMA CULTURES
547
described by Engvall and Perlmann ~°is rapidly becoming the immunoassay method of choice. The CIEIA described here is a modification of the original ELISA procedure which allows quantification of Ig down to the 10 ng/ml level. Several practical aspects of the CIEIA require discussion. At what point in the screening of hybridomas would the CIEIA be used? Since uncloned hybridoma cultures often contain multiple clones secreting the same Ig isotype or subclass, the CIEIA would be of little value. It is best to initially screen the cultures for antibody to the desired antigen by standard methods, choosing positives for subsequent cloning. When analyzing clones, they should be screened for both specific antibody and total Ig. By determining the relative cell number (clone size, degree of confluence of the microculture), the cultures with clones secreting the most Ig can be identified. Of course the quantification can be expressed as molecules of IG secreted per cell per minute if the cells are actually counted and sampling done over time. It is essential that the reagents employed have heavy chain specificity, particularly if more than one isotype of Ig is present in the cultures. For instance, in cultures with both IgG and IgA antibodies, in which both isotypes bear h light chains, absolute quantification of either antibody would be impossible. Obviously the purity of the solid phase Ig would be equally essential. The CIEIA is quantitative and reproducible, and can be performed in less than 3 hr. Its increased precision makes it more attractive than the ACEIA method. It is essential to remember that prior to employing the CIEIA in experimental studies, each reagent must be optimized for concentration with the goal of creating a standard curve that is linear and as steep as possible. Acknowledgments We thank Susan McLaughlin for editorial assistance, and the U.S. Army Chemical Research and Development Center for support.
~0 E. Engvall and P. Perlmann,
Immunochemistry 8, 871 (1971).
ANTIBODY
MEASUREMENT
IN H Y B R I D O M A C U L T U R E S
541
The EIFA described here has several distinct advantages over other assay systems, such as the RIA. When read visually, this EIFA takes no more than 2 hr to complete. These filter plates are more sensitive than equivalent poly(vinyl chloride) plate assays, they do not require a lengthy pretreatment of the assay plate in order to reduce nonspecific adsorption of antibody, and washes can be completed in less than 1 min without centrifugation, or poly-L-lysine or glutaraldehyde fixation which may alter epitopes. Moreover, cells are not lost or reduced as in the centrifugationwash steps of other assays. Acknowledgment We would like to acknowledge the support of NIH Grants EY-0393 and CA-32047.
[51] M e a s u r e m e n t of M o n o c l o n a l I m m u n o g l o b u l i n C o n c e n t r a t i o n s in H y b r i d o m a C u l t u r e s b y C o m p e t i t i v e Inhibition E n z y m e I m m u n o a s s a y
By
KENNETH W. HUNTER, JR. a n d JOHN
M. BOSWORTH, JR.
The selection of hybridomas most often involves a determination of specificity, and less often affinity. Still less often addressed is the secretion rate of monoclonal antibodies by particular hybridoma clones. This latter issue may be of secondary but practical importance as monoclonal antibodies are further commercialized. Although mouse monoclonal antibodies can be produced in large scale by the ascites method, human monoclonal antibody production presently relies on mass in vitro culture. By selecting clones that exhibit the highest rates of antibody secretion, one can substantially improve the yield per volume of culture. This chapter describes a competitive inhibition enzyme immunoassay1 for accurately measuring the levels of immunoglobulin encountered in hybridoma cultures, the parameters that govern performance of the assay, and the advantages of the method over antigen-capture enzyme immunoassay. Materials and Methods
Reagents. Alkaline phosphatase-conjugated goat anti-rabbit IgG and p-nitrophenyl phosphate were obtained from Sigma Chemical Co. (St. t j. M. Bosworth, A. A. Brimfield, J. A. Naylor, and K. W. Hunter, J. Immunol. Methods 62, 331 (1983).
METHODS IN ENZYMOLOGY, VOL. 121
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
542
MONOCLONAL ANTIBODIES
[51]
Louis, MO). Rabbit antibodies to mouse IgGi (Lot RFC028), and the myeloma protein MOPC 21 (IgG~, Lot DG071) were obtained from Litton Bionetics (Kensington, MD). Human IgG (Lot 25), goat anti-human IgG (Lot BD1 l-B), and alkaline phosphatase-conjugated rabbit anti-goat IgG were obtained from Kirkegaard & Perry Laboratories (Rockville, MD). Competitive Inhibition Enzyme Immunoassay (CIEIA). The solidphase adsorbant used in the CIEIA was a 96-well round-bottomed polystyrene microtiter plate (Dynatech Laboratories, Alexandria, VA). It should be noted that some plastic microtiter plates work very poorly in the CIEIA. The critical importance of reagent concentrations on the performance of the CIEIA will be illustrated in the experimental section. Each well of the microtiter plate is incubated with 50/zl of an optimal concentration of the immunoglobulin (Ig) to be quantified (e.g., mouse IgGl), diluted in 0.1 M NaHCO3 buffer (pH 9.6). The time and temperature of the incubation are not critical; we routinely use overnight incubations at 4°. The fluid phase of the competitive inhibition reaction is set up in a separate microtiter plate. We recommend washing this plate with phosphate-buffered saline containing 0.5% polyoxyethylene sorbitan monolaurate (PBS-T) to reduce adsorption of reagents to the plastic. The fluid-phase reaction consists of equal volumes (50/zl) of a known standard concentration of Ig (or your sample that contains an unknown concentration) and an antibody to that Ig. Routinely, for generating the standard curve we make serial log dilutions of Ig in the same vehicle as the test sample (e.g., culture medium), beginning at 100/zg/ml. The anti-Ig is used in a concentration that does not saturate the binding sites of an Ig-coated microwell. The mixtures are incubated for 1 hr at 4°. The Ig-coated plates are washed by five cycles of filling and emptying all wells with cold PBS-T. Aliquots of 50/zl of the Ig-anti-Ig mixtures are transferred to the Ig-coated plates and incubated for 30 min at 4 °. The Ig in the fluid phase competes with the solid-phase-bound Ig for the anti-Ig. The higher the concentration of Ig in the fluid phase the less anti-Ig will bind to the solid phase. The amount of solid-phase-bound anti-Ig is detected by an enzyme-tagged second antibody. Although reasonable results can be obtained with an enzyme-labeled first antibody, we have found that the double antibody method provides an amplified signal. After washing as described above, 50 /zl of an optimal PBS-T dilution of an alkaline phosphatase-conjugated antibody to the anti-Ig is added for another 30 min at 4°. Finally, the plates are washed and 100/zl of substrate (1 mg/ml, p-nitrophenyl phosphate in 0.1 M ethanolamine buffer, pH 9.6) is added to each well. The enzymatic reaction is allowed to proceed for 30 min at room temperature, and then the colored product in each well is measured spectrophotometrically at 405 nm with a Titertek Multiscan
[51]
ANTIBODY MEASUREMENT IN HYBRIDOMA CULTURES
543
micro-ELISA reader (Flow Laboratories, Vienna, VA). The absorbance is inversely related to the concentration of Ig in the fluid phase, and a standard curve is prepared by plotting Ig concentration versus absorbance. Each point on the standard curve represents the average absorbance of six replicates. Maximum absorbance is that obtained by incubating only vehicle with anti-Ig. Linear regression analysis of these data usually yield correlation coefficients greater than 0.90. To determine the Ig concentration of a test sample, the mean absorbance value of the test sample (hybridoma culture supernatant) is entered as the y value in the regression equation of the standard curve, and the corresponding x value is calculated. This analysis system eliminates the subjectivity inherent in deriving data values from manually plotted standard curves, and also rectifies the small deviation from linearity seen in most standard curves. Antigen-Capture Enzyme Immunoassay (ACEIA). The solid-phase adsorbant and washing procedure were the same as used in the CIEIA. Plates were coated overnight at 4 ° with 50/xl of an optimal dilution of antiIg in 0.1 M NaHCO3 buffer (pH 9.6). A standard curve was generated by incubating serial log dilutions (50/xl) of Ig standard in anti-Ig-coated microtiter plates for 30 min at 4°. After washing, the "captured" Ig was sandwiched by the addition of the same anti-Ig in PBS-T for 30 min at 4 °. The remainder of the ACEIA is exactly the same as that described for the CIEIA. Optimization of the CIEIA. To illustrate the critical importance of optimization of reagent concentrations in the performance of the CIEIA, an assay of human IgG will be used. In this example, purified human IgG was adsorbed to the solid phase, and a goat anti-human IgG along with the same purified human IgG were the fluid-phase reagents. The solid-phase binding was developed with a rabbit anti-goat lgG that was tagged with alkaline phosphatase. Reaction conditions were as described in the Materials and Methods section. In this optimization experiment, microtiter plates were coated with 10, 1, or 0.1 /zg human IgG/ml. Four different concentrations of goat antihuman IgG (10, 5, 1, 0.1/xg/ml) and rabbit anti-goat IgG (10, 2, 1, 0.2/zg/ ml) were also tested. The results of this optimization experiment are shown in Fig. 1. Each reagent concentration was critical to the CIEIA. As we described in an earlier paper, j the slope of the standard curve should be as steep as possible so that variability in absorbance values plotted arithmetically on the y axis will not yield unwieldly variabilities in Ig concentrations plotted logarithmically on the x axis. It can be seen in Fig. 1 that a human IgG coating concentration of 10/zg/ml yielded the highest absolute absorbance values, but the slopes of the curves were quite fiat
544
MONOCLONAL ANTIBODIES
1.5 g --~ 1.(1
j
[51]
b:
g
,,.,~
c_
c~ , . 4 ~
b
'~ 0.5 ,,,7,
Jia:illLilll
~
II~P--4P'~r.~k-IP"~ld
E ~
F
G
aH
I
J
K
L
E 1.5 g
----1.0 o
'~ 0.~
E_1.5 g
~0.5 ,
t
1000 10 0,1 0.001 1000 10 0.1 0.001 1000 10 0.1 0.001 1000 10 0.1 0,001
[pg/ml] FIG. 1. Optimization of reagent concentrations for CIEIA of human IgG. The coating concentrations of human IgG were 10/.tg/ml (panels A-D), 1 /~g/ml (panels E-H), and 0.1 /~g/ml (panels I-L). Goat anti-human IgG concentrations were 10/xg/ml (panels A, E, and I), 5/zg/ml (panels B, F, and J), 1 p.g/ml (panels C, G, and K), and 0.1 p~g/ml (panels D, H, and L). Rabbit anti-goat IgG concentrations were 10/xg/ml (a), 5/zg/ml (b), 2/xg/ml (c), and 0.2 tzg/ml (d). Each point is the mean of six replicates.
[51]
ANTIBODY MEASUREMENT IN HYBRIDOMA CULTURES
545
regardless of the concentrations of the other reagents. When plates were coated with 0.1/zg/ml, the absolute absorbance values were substantially lower, and no combination of reagents produced a reasonable standard curve. At what would be considered the optimal human IgG coating concentration (1 /xg/ml), standard curves with the desired characteristics were generated. However, as can easily be seen the concentrations of goat anti-human IgG (panels E - H ) and rabbit anti-goat IgG (curves a-d on each panel) played a critical role. It appeared that with these particular reagents, 1 or 5/xg of goat anti-human IgG per milliliter coupled only with the highest concentration of rabbit anti-goat IgG (10/xg/ml) yielded good standard curves. These data point out the importance of optimization experiments in the development of CIEIA methods. Comparison of CIEIA and ACEIA. Another method for quantifying immunoglobulins is ACEIA, or sandwich immunoassay. We have compared this method with CIEIA and find the latter to be superior. For example, in Fig. 2 we show standard curves obtained for mouse IgGj 1.4
1.2
|
1.0
0.8 oo
0.6
0.4
0.2
I
I
100
10
I
I
I
I
I
1 0.1 0.01 0,001 0.0001 IgG1 (pg~ml)
FIG. 2. Comparison of standard curves for mouse monoclonal IgG~ concentrations by CIEIA (0) and ACEIA (O). Each point is the mean -+ one standard deviation of six replicates.
546
MONOCLONAL ANTIBODIES
[51]
under optimized conditions (for both CIEIA and ACEIA). In both assay modes, linear curves from approximately 100 /xg/ml to 10 ng/ml were obtained. Though similar in sensitivity, the curves had very different slopes; the CIEIA curve was steeper than the ACEIA curve. Since both methods had been optimized with respect to reagent concentrations, it is likely that the difference in slope is a function of the mode of assay. This difference in slope resulted in an average standard deviation from the mean of each point on the ACEIA curve of 0.06 absorbance units, compared to only 0.02 for the CIEIA. Data from several experiments indicated excellent reproducibility in standard curves, and confirmed the larger variability associated with each point on the ACEIA curve. Together, the steeper slope and lower variability of the CIEIA curve make this assay method superior to the ACEIA. As an illustration, if the variability of IgG1 concentrations at the point on Fig. 2 where the curves intersect (0.95 absorbance units and 0.9/~g/ml IgG0 is assessed, the range of one standard deviation above and below the mean is 0.02-0.33/zg/ml for ACEIA, but only 0.08-0.11/xg/ml for CIEIA. Therefore, the precision with which IgG1 concentrations can be measured is greater in the CIEIA. Discussion The capability of accurately measuring the rate of monoclonal antibody secretion by hybridoma cultures should facilitate the selection of clones with the greatest production potential. The concentrations of Ig found in hybridoma cultures produced in our laboratory range from 25/~g/ ml to I ng/ml, with an average of approximately 1 /xg/ml. Probably the most common method for quantifying Ig is by single radial immunodiffusion, 2,3 but this method cannot detect the low levels found in hybridoma supernatants. Other techniques which have been employed to detect Ig in cultures include immunoelectrophoresis, 4,5 hemagglutination inhibition, 6 and reverse plaquing, 7 all of which are semiquantitative. Radioimmunoassay for Ig is sensitive and quantitative, but because it requires the use of isotopes, 8,9 the enzyme-linked immunosorbent assay (ELISA) originally 2 G. Mancini, A. O. Carbonara, and J. F. Heremans, lmmunochemistry 2, 235 (1965). 3 j._p. Vaerman, this series, Vol. 73, Part B, p. 291. 4 j. L. Fahey, I. Finegold, A. S. Rabson, and R. A. Manaker, Science 152, 1259, 1966. 5 R. van Furth, H. R. E. Schuit, and W. Hijmans, Immunology 11, 1 (1966). 6 F. P. Siegal and M. Siegal, J. lmmunol. 118, 642 (1977). 7 G. A. Molinaro and S. Dray, Nature (London) 248, 515 (1974). 8 T. Hirano, T. Kuriani, T. Kishimoto, and Y. Yamamura, J. Immunol. 119, 1235 (1977). 9 S. M. McLachland, B. R. Smith, and R. Hall, J. Immunol. Methods 21, 212 (1970).
[51]
ANTIBODY MEASUREMENT IN HYBRIDOMA CULTURES
547
described by Engvall and Perlmann ~°is rapidly becoming the immunoassay method of choice. The CIEIA described here is a modification of the original ELISA procedure which allows quantification of Ig down to the 10 ng/ml level. Several practical aspects of the CIEIA require discussion. At what point in the screening of hybridomas would the CIEIA be used? Since uncloned hybridoma cultures often contain multiple clones secreting the same Ig isotype or subclass, the CIEIA would be of little value. It is best to initially screen the cultures for antibody to the desired antigen by standard methods, choosing positives for subsequent cloning. When analyzing clones, they should be screened for both specific antibody and total Ig. By determining the relative cell number (clone size, degree of confluence of the microculture), the cultures with clones secreting the most Ig can be identified. Of course the quantification can be expressed as molecules of IG secreted per cell per minute if the cells are actually counted and sampling done over time. It is essential that the reagents employed have heavy chain specificity, particularly if more than one isotype of Ig is present in the cultures. For instance, in cultures with both IgG and IgA antibodies, in which both isotypes bear h light chains, absolute quantification of either antibody would be impossible. Obviously the purity of the solid phase Ig would be equally essential. The CIEIA is quantitative and reproducible, and can be performed in less than 3 hr. Its increased precision makes it more attractive than the ACEIA method. It is essential to remember that prior to employing the CIEIA in experimental studies, each reagent must be optimized for concentration with the goal of creating a standard curve that is linear and as steep as possible. Acknowledgments We thank Susan McLaughlin for editorial assistance, and the U.S. Army Chemical Research and Development Center for support.
~0 E. Engvall and P. Perlmann,
Immunochemistry 8, 871 (1971).