An ELISA for screening hybridoma cultures for monoclonal antibodies against a detergent solubilized integral membrane protein

Journal oflmmunologicalMethods, 75 (1984) 141-148

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Elsevier JIM03299

An ELISA for Screening Hybridoma Cultures for Monoclonal Antibodies against a Detergent Solubilized Integral Membrane Protein William D. N o t e b o o m 1 , , K i m E. Knurr 2 Haeng S. Kim 2 Willis G. R i c h m o n d 2 A r l e n e P. M a r t i n 1,2 a n d Marie L. V o r b e c k 1,2 Departments of ~ Biochemistry and 2 Pathology, University of Missouri-Columbia, School of Medicine, Columbia, M O 65212, U.S.A.

(Received 23 March 1984, accepted 31 August 1984)

A method is described for the binding of a detergent solubilized integral membrane protein to polystyrene immunoassay plates. Addition of Bouin's fluid, a histochemical fixative, to wells of plates containing the detergent solubilized antigen, followed by low speed centrifugation, is sufficient to promote binding of antigen in the presence of Triton X-100 concentrations as high as 1.75%. The binding of antigen is rapid and the entire binding procedure, including removal of fixative and washing of the plates, can be accomplished in less than 15 min. Immunological specificity of the bound antigen is retained. This method has been used to effectively screen hybridoma cultures for specific antibodies. Key words: E L I S A - monoclonal antibody - antigen binding - detergent solubilized membrane protein cytochrome oxidase

Introduction

Antibody producing cells can be immortalized by fusion with myeloma cells using the techniques of Krhler and Milstein (1976). Although the hybrids formed can produce large amounts of monoclonal antibody, a rapid, simple, and sensitive screening assay to determine the specificity of the cloned immunoglobulins facilitates the production of a specific monoclonal antibody. The most frequently used techniques for screening monoclonal antibodies produced by hybridomas are based on the observation of Cart and Tregear (1967) that proteins bind tightly to plastic surfaces. The ease with which most soluble antigens may be insolubifized by simple adsorption to plastic 96-well microtiter plates has made this method very popular. Detection of monoclonal antibodies to membrane associated proteins presents a * To whom correspondence should be addressed. 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.

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special problem because the surfactant effects of non-ionic detergents used to solubilize membrane antigens could retard or prevent protein binding to polystyrene microtiter wells (Newman et al., 1981; Palfree and Elliott, 1982). In the process of preparing monoclonal antibodies against Triton X-100 solubilized rat liver cytochrome oxidase, we developed a modified ELISA technique which is rapid, simple, and sensitive for screening the many culture supernates generated during production and cloning of hybridomas.

Materials and Methods

Preparation of antigen A partially purified cytochrome oxidase preparation was used as the coating antigen. This preparation was solubilized from Triton X-114 treated rat liver submitochondrial particles (Rascati and Parsons, 1979) essentially as described by Jacobs et al. (1966), using 50 mM potassium phosphate, pH 7.4, containing 5% Triton X-100. The Triton X-100 extract was essentially free of other hemoproteins and contained 5.5 to 6 nmol cytochrome a a 3 / m g protein. A highly purified preparation of cytochrome oxidase (15-16 nmol cytochrome a a 3 / m g protein), obtained by chromatography of the Triton X-100 extract on DEAE-Sepharose was used for immunization of B A L B / c mice. Cytochrome aa 3 concentration was determined by difference spectroscopy (Vorbeck et al., 1982); protein was determined fluorimetrically using Fluram® (Vorbeck et al., unpublished).

Preparation of antisera Female B A L B / c mice, weighing approximately 16 g, were given 5 intraperitoneal injections of the highly purified cytochrome oxidase in Freund's incomplete adjuvant at weekly intervals. The mice were bled after the fourth and fifth immunizations to obtain hyperimmune serum to be used for the development of the ELISA and as the positive control for screening the hybridoma culture supernates. Preimmune serum was obtained from the mice prior to the first immunization and was used as the negative control. The IgG concentration of the mouse serum and the monoclonal antibody preparations was determined by rocket immunoelectrophoresis (Laurell, 1972) using mouse IgG (Sigma Chemical Co., St. Louis, MO) as standard.

Monoclonal antibodies Monoclonal antibodies were derived from hybridomas obtained from the fusion of the mouse SP2/0-Ag14 plasmacytoma cell line with spleen cells from B A L B / c mice immunized with a highly purified preparation of rat liver cytochrome oxidase. The preparation and characterization of the hybridoma cell lines will be described elsewhere.

Preparation of microtiter plates Partially purified cytochrome oxidase was diluted with phosphate buffered saline (PBS), pH 7.2, to 40 /~g protein per ml, and contained 0.06% Triton X-100. Fifty

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microliters of the diluted antigen were added to each well of Nunc-Immunoplate II polystyrene microtiter plates. Bouin's fluid, 200/~1, was then added to each well and the plates were centrifuged at 500 x g for 7 min. The Bouin's fluid was removed by aspiration and the wells were washed twice with 50% ethanol and once with PBS. Non-specific binding sites were blocked by the addition of 275 /~1 of 3% bovine serum albumin (BSA) in PBS containing 0.01% thimerosal. Plates were allowed to stand at room temperature for 1 h. If the plates were to be used at a later date, the plates containing the blocking solution were covered with pressure film and stored in the dark at 4°C. Coated plates could be stored for up to a week. The blocking solution was removed from all plates by aspiration and the wells were washed twice with PBS prior to the addition of the primary antibody.

ELISA procedure Immediately prior to an assay, 100 /zl of the preimmune serum, hyperimmune serum or hybridoma culture supernate were added to each well. Sera were diluted 1 : 100 while the hybridoma culture supernate was added undiluted. The plates were incubated at 37°C for 1 h. The test solutions were removed by aspiration and the wells were washed 4 times with PBS containing 0.05% Tween 20. To each well was added 50 ~tl of a 1 : 2000 dilution of goat anti-mouse I g G conjugated with horseradish peroxidase in PBS. The plates were incubated for 1 h at 37°C. The secondary antibody conjugate was removed by aspiration and the wells were washed 6 times with PBS containing 0.05% Tween 20. To each well was added 200 ~tl of substrate solution containing 3.0% hydrogen peroxide and 4.0 mM 2,2'-azino-di-(3-ethyl benzthiazoline sulfonic acid) diammonium salt (ABTS) in McIlvaine's citric acidphosphate buffer, p H 4.0. The plates were then incubated at 37°C in the dark for 15 min. At the end of the incubation, 40 ~tl of 10% sodium dodecyl sulfate were added to each well and the contents of the wells were mixed by gentle agitation of the plates. The absorbance was determined at 405 nm using a BioTek microplate reader. Wells treated with preimmune serum were used as negative controls and provided a measure of non-specific reagent binding. The absorbance value obtained by subtracting the absorbance of the negative control was used as a measure of antigen binding to the microtiter plates. An absorbance value which was twice that of the negative control was considered a positive result when screening for antibody producing hybridomas. ELISA competition assay The specificity of the ELISA procedure was determined by an ELISA competition assay. The unrelated antigens used in these experiments were a Triton X-100 extract of purified rat liver plasma membranes and BSA. Rat liver plasma membranes were isolated and purified according to Neville (1968) as modified by Ray (1970). In the competition assays 20 /~1 of the Triton X-100 extract of rat liver plasma membranes (2 mg protein/ml), BSA in 0.05% Triton X-100 (2 mg p r o t e i n / ml), or 0.05% Triton X-100 were mixed with 100 /~1 of each dilution of anticytochrome oxidase monoclonal antibody and preincubated at 37°C for 30 min. The ELISA procedure was carried out as described above.

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Results

An ELISA for screening hybridoma supernates for monoclonal antibodies against detergent solubilized integral membrane proteins requires effective binding of the membrane protein on a solid phase in the presence of detergent. The effectiveness of Bouin's fluid, a histochemical fixative, in facilitating the binding of Triton X-100 solubilized rat liver cytochrome oxidase to polystyrene microtiter plates was evaluated using a sandwich technique with mouse hyperimmune serum against a highly purified preparation of rat liver cytochrome oxidase as the primary antibody. Horseradish peroxidase conjugated goat anti-mouse IgG was the secondary antibody. Two-fold serial dilutions of partially purified cytochrome oxidase in PBS were used for coating of the plates. The effect of Bouin's fluid on the final ELISA signal is given in Table 1. Bouin's fluid increased the binding of Triton X-100 solubilized cytochrome oxidase at least 2-fold over the entire concentration range used (0.1 /~g to 12.8 /tg). Non-specific binding of the horseradish peroxidase conjugated goat anti-mouse IgG was observed with the negative serum control in the absence of Bouin's fluid. In the presence of Bouin's fluid, however, non-specific binding was decreased and accounted for less than 25% of the total absorbance (data not shown). The mechanism by which Bouin's fluid effects the increased binding probably involves denaturation and/or precipitation of the Triton X-100 solubilized cytochrome oxidase. A centrifugation step was included to facilitate maximum binding to the microtiter wells. If the centrifugation step was omitted, cytochrome oxidase binding, especially at low concentrations, was variable even in the presence of Bouin's fluid. Optimal time for color development by the chromogen-substrate solution was determined by varying the incubation time over a period of 60 min. Microtiter plates coated with cytochrome oxidase (2 btg/well) using Bouin's fluid were incubated with TABL E I E F F E C T OF BOUIN'S F L U I D ON B I N D I N G OF T R I T O N X-100 SOLUBIL1ZED C Y T O C H R O M E OXIDASE TO P O L Y S T Y R E N E M I C R O T I T E R PLATES Values shown represent the mean and standard deviation of triplicate samples. Cytochrome oxidase (/Lg/well)

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0.174 + 0.022 0.207 _+0.048 0.284 _+0.028 0.297 _+0.044 0.338 _+0.041 0.352 4- 0.041 0.501-4- 0.043 0.530 ± 0.018

0.436 _+0.029 0.561 _+0.016 0.579 _+0.030 0.663 _+0.031 0.823 _+0.039 0.923 ± 0.046 1.144 ± 0.018 1.034 + 0.014

a Minus the absorbance of the negative control.

145 a 1:100 dilution of hyperimmune serum followed by the horseradish peroxidase conjugated second antibody. The wells were washed as described in Materials and Methods and the chromogen-substrate solution was added. Incubation conditions were maintained constant and exposure of the plates to light was minimal. As shown in Fig. 1, the absorbance of the positive control increased rapidly during the first 15 min of incubation and thereafter remained constant for up to 60 min of incubation. With the negative control serum, absorbance increased to about 0.2 after 10 min incubation with further increase to an absorbance of approximately 0.4 by 60 min. The difference between the absorbance of the positive control and the negative control was maximum following 15 min incubation. Thus, subsequent studies were performed using an incubation time of 15 min with the chromogen-substrate solution. The effect of varying concentrations of Triton X-100 on cytochrome oxidase binding in the presence and absence of Bouin's fluid is shown in Fig. 2. In the presence of Bouin's fluid, cytochrome oxidase binding was unaffected by a Triton X-100 concentration range of 0.06 to 1.75%. In marked contrast, in the absence of Bouin's fluid there was only partial binding of cytochrome oxidase at low concentrations of Triton X-100 (0.06 and 0.09%) and at concentrations of 0.23% or greater, Triton X-100 abolished virtually all binding. To determine the sensitivity of the modified ELISA in detecting antibody, microtiter plates were coated with a constant concentration of the partially purified cytochrome oxidase (2 ~g/well) using Bouin's fluid and centrifugation. After washing and blocking of non-specific binding sites, 100 ~1 of 2-fold serial dilutions of mouse hyperimmune serum were added. The plates were processed further as described in Materials and Methods. Fig. 3 shows the dependence of absorbance on the specific binding of mouse anti-cytochrome oxidase immunoglobulin. At the

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lowest level of immunoglobulin measured, 0.077/xg/well, the change in absorbance was easily detectable. The specificity of the ELISA was assessed by determining the ability of unrelated antigens to compete with the solubilized and immobilized cytochrome oxidase for binding of a monoclonal antibody to cytochrome oxidase. As shown in Fig. 4, preincubation of an anticytochrome oxidase monoclonal antibody with either a Triton X-100 extract of rat liver plasma membranes or bovine serum albumin has essentially no effect on the ability of the monoclonal antibody to bind to the immobilized cytochrome oxidase over the range of monoclonal antibody of 0.045 to 0,5

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0.735 /~g IgG/well. The sensitivity demonstrated in this experiment was less than 0.050 #g per well for the detection of antibody in culture fluid and this is in close agreement with the limit of detection of antibody in hyperimmune serum. The dependence of the final ELISA signal on the concentration of partially purified cytochrome oxidase used for coating the microtiter plates was determined using a 1 : 3 2 0 0 dilution of monoclonal antibody against cytochrome oxidase. As shown in Fig. 5, an excellent linear correlation (r = 0.983) was obtained over a

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148 c o n c e n t r a t i o n range of 0.125 to 2.0 ~g of p a r t i a l l y purified c y t o c h r o m e oxidase per well.

Discussion T h e i m m o b i l i z a t i o n of soluble p r o t e i n antigens on m i c r o t i t e r plates for E L I S A techniques is a time c o n s u m i n g process. The use of integral m e m b r a n e p r o t e i n s as antigens presents special p r o b l e m s because of the s u r f a c t a n t effects of the detergents used for solubilization. Several techniques have been r e p o r t e d to facilitate antigen b i n d i n g to a solid phase in the presence of detergent. Eash and Villarejo (1983) used d e s i c c a t i o n at r o o m t e m p e r a t u r e to p r o m o t e b i n d i n g of Escherichia coli m e m b r a n e f r a g m e n t s or lactose p e r m e a s e to microtiter plates. Palfree a n d Elliott (1982) used nitrocellulose m e m b r a n e s as a solid phase for Ia g l y c o p r o t e i n s solubilized with deoxycholate. A l t h o u g h this p r o c e d u r e was suitable for a n u m b e r of detergents, T w e e n 80 or T r i t o n X-100 which are used widely to solubilize integral m e m b r a n e p r o t e i n s h a d adverse effects on antigen b i n d i n g unless c o n c e n t r a t i o n s of less than 0.01% were used. In the present work, we have d e s c r i b e d a m o d i f i e d E L I S A for T r i t o n X-100 solubilized c y t o c h r o m e oxidase which uses Bouin's fluid to facilitate antigen b i n d i n g to the microtiter plates. A n t i g e n - a n t i b o d y i n t e r a c t i o n does not a p p e a r to be affected b y the use of Bouin's fluid. The assay is sensitive and specific; c y t o c h r o m e oxidase b i n d i n g was unaffected b y T r i t o n X-100 c o n c e n t r a t i o n s as high as 1.75%. This, together with the s h o r t e n e d time required for antigen b i n d i n g (15 min), m a k e s the p r o c e d u r e a p p l i c a b l e for other m e m b r a n e p r o t e i n s which must be h a n d l e d in d e t e r g e n t solutions.

Acknowledgments This w o r k was s u p p o r t e d b y N I H G r a n t A G - 0 0 5 9 0 from the D e p a r t m e n t of H e a l t h a n d H u m a n Services.

References Catt, K. and G.W. Tregear, 1967, Science 158, 1570. Eash, J. and M.R. Villarejo, 1983, Arch. Biochem. Biophys. 220, 495. Jacobs, E.E., E.C. Andrews, W. Cunningham and F.L. Crane, 1966, Biochem. Biophys. Res. Commun. 25, 87. K6hler, G. and C. Milstein, 1976, Eur. J. Immunol. 6, 511. LaureU, C.-B., 1972, Scand. J. Clin. Lab. Invest. 29 (suppl. 124), 21. Neville, Jr., D.M., 1968, Biochim. Biophys. Acta 154, 540. Newman, P.J., R.A. Kohn and A. Hines, 1981, J. Cell Biol. 90, 249. Palfree, R.G.E. and B.E. Elliott, 1982, J. Immunol. Methods 52, 395. Rascati, R.J. and P. Parsons, 1979, J. Biol. Chem. 254, 1586. Ray, T.K., 1970, Biochim. Biophys. Acta 196, 1. Vorbeck, M.L., A.P. Martin, J.K.J. Park and J.F. Townsend, 1982, Arch. Biochem. Biophys. 214, 67.