Journal of Immunological Methods, 139 (1991) 55-64
55
© 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100160A
JIM 05912
Stability of monoclonal antibody-defined epitopes * Byron E. Wilson, Susan Sun, M e h m e t O z t u r k a n d Jack R. W a n d s Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, Charlestown, MA 02129, U.S.A., and Department of Medicine, Harvard Medical School, Boston, MA 02114, U.S.A. (Received 28 December 1990, accepted 15 January 1991)
Epitope instability can limit the applications of monoclonal antibody (mAb) technology in laboratory and clinical research. We exposed a group of representative antigens on human hepatocellular carcinoma (HCC) cells to physiochemical insults to study epitope stability as measured by mAb immunoreactivity. Each epitope was found to have a unique pattern of instability which serves to biophysically characterize the antigen and de'fines the conditions to which the antigen can be exposed during laboratory and clinical investigations. Individual antigens were found to be unstable within a surprisingly well defined window of solvent polarities while being stable on either side of that window. Several antigens were observed to be unstable when exposed to transient changes in pH. When a critical temperature between 42 ° C and 65 °C was achieved, epitopes which were thermosensitive underwent a sudden loss in immunoreactivity. This critical temperature was found to be pH dependent. The effects of polarity, pH, and temperature on epitope stability are consistent with changes in protein structural conformation. In addition, we found that certain fixatives cause a time and concentration dependent loss of epitope immunoreactivity. This study provides a rapid and easy determination of monoclonal antibody-defined epitope stability; the results of which serve to guide further studies on the antigen and to characterize the antigen on the basis of its unique physiochemical stability. Key words." Cell surface antigen; Epitope; Monoclonal antibody
Introduction
The development of monoclonal antibody (mAb) technology has led to a new generation of
Correspondence to: J.R. Wands, Molecular Hepatology Laboratory, M G H Cancer Center, 7th Floor, M G H East, 149 13th Street, Charlestown, MA 02129, U.S.A. * This work was supported in part by Grants CA-3571, AA02666, AA-01862 and HD-20469 from the National Institutes of Health and a grant from Association pour la Recherche sur le Cancer, Villejuif, France. J.R.W. is the recipient of a Research Scientist Award AA-00048.
research and clinical reagents. Morloclonal antibodies are now instrumental in antigen identification, characterization, and purification; in cloning of translated genetic products; and in immunohistology for investigative and diagnostic purposes (Gatter et al., 1984; Tainer et al., 1984; DeLellis et al., 1987; Takahashi et al., 1988, 1989a, b; Ozturk et al., 1989). In addition, monoclonal antibodies are becoming increasing useful as potential in vivo immunotargeting and therapeutic reagents. (Vitetta et al., 1987; Takahashi et al., 1988, 1989a, b). cad The advantage of monoclonal over polyclonal antibodies is derived from the exquisite specificity
56 afforded by single epitope recognition. However, the potential applications of a given mAb can be limited if the epitope recognized by that antibody is unstable. For example, immunohistological techniques often require extensive processing of tissue specimens for preservation of morphology; Western immunoblotting requires antigen solubilization and often denaturation; protein purification may involve acidic and alkaline conditions, use of polar and nonpolar solvents, or thermal precipitation. Under such non-physiological conditions, a given epitope's immunoreactivity may be destroyed or substantially altered. Polyclonal antibodies often recognize a spectrum of many different epitopes such that physiochemical modifications destroy only a subset of epitopes, resulting in a lowered, but generally not completely lost, total immunoreactivity (Vita et al., 1985; DeLellis et al., 1987). In contrast, as a single epitope system, the immunoreactivity of an antigen to a monoclonal antibody may be completely lost following a physiochemical insult (Hancock et al., 1982; Garter et al., 1984; Oda et al., 1984; Ciocca et al., 1986; DeLellis et al., 1987; Pollard et al., 1987). Determining the stability of a given epitope provides a partial characterization of the antigen. For example, changes in immunoreactivity following physiochemical insult have been used to differentiate between (and define the structural basis for) rheumatoid factor crossidiotypes (Agnello et al., 1986); to detect subtle differences in type IV collagen between tissues (Linsenmayer et al., 1984); to study the conformational substructure of thermolysin (Vita et al., 1985); and to study the relationship between structure and function for IgE domains containing the mast cell receptor binding site (Dorrington et al., 1973; Hastings et al., 1988). On a more practical level, determining the stability of a given epitope delineates the conditions necessary for use of the monoclonal antibody as an investigative or clinical tool (Hancock et al., 1982; Oda et al., 1984; Pollard et al., 1987). We have developed methods by which one can readily determine the stability of epitopes to various physiochemical conditions often encountered in laboratory and clinical research, and have applied these methods to a panel of well defined tumor-associated antigens.
Materials and methods
Cell culture The hepatocellular carcinoma cell line FOCUS developed by us (Lun et al., 1984) was maintained in Eagle's minimum essential medium (M.A. Bioproducts, Walkersville, MD) supplemented with 10 /~M of nonessential amino acids and 10% fetal bovine serum inactivated at 56°C. Immunizing cells were grown from the second passage of the FOCUS cell line and were harvested from monolayer culture with EDTA/Versene in the absence of trypsin, then washed with PBS. Monoclonal antibody production 6-week-old B A L B / c female mice were first immunized by intraperitoneal injection of 4.0 x 10 6 FOCUS cells emulsified in 50% (v/v) complete Freund's adjuvant. Secondary immunizations were performed 6-10 weeks later by intravenous injection of 4.0 × 106 intact cells in 0.2 ml of PBS. 3 days later, mouse splenocytes were fused with either SP20, NS1, or X63 mouse myeloma cell lines with 30% (w/v) polyethylene glycol. Resulting hybridomas were maintained and selected as described (Laemmli et al., 1970). Screening for mAbs was performed by the indirect binding assay as described (Wilson et al., 1988). Purification and iodination of mAbs were performed as described (Takahashi et al., 1989; Ey et al., 1990). Properties of human tumor associated antigens We have selected six anti-FOCUS monoclonal antibodies, named AF5, AF20, FB50, SF25, SF90 and XF8, for use in this study. All these mAbs recognize antigens which share the common feature of having a high level of expression in hepatocellular carcinoma (HCC) with little or no expression in adjacent uninvolved liver (Wilson et al., 1988; unpublished results). Three antibodies (AF5, FB50, and SF90) recognize proteins identifiable by Western immunoblotting. Furthermore these mAbs are known to recognize epitopes contained within the primary structure of the protein. The antigen recognized by AF5 is a 36 kDa phosphoprotein, which in addition to HCC tissue, has been found on all transformed cell lines tested (Frohlich et al., 1990). The
57 mAb FB50 recognizes two families of 50 kDa and 100 kDa proteins (data not shown). SF90 recognizes a 50 kDa protein distinct from that recognized by FB50 (Ozturk et al., 1989). In addition to high HCC expression, the FB50 and SF90 antigens are highly expressed in normal adrenal glands but are expressed at considerably lower levels in other normal human tissues. The SF90 antigen has been present on all malignant human cells thus far tested. AF20, SF25, and XF8 recognize antigens highly expressed in adenocarcinomas of the colon, lung and liver and most other malignant tumors, with little if any expression on normal human tissues (Takahashi et al., 1988, 1989a). None of these three antigens are demonstrable by Western immunoblotting. All three antigens have been successful in imaging tumors in nude mouse models (Takahashi et al., 1988, 1989a, b). Although none of these antigens are demonstrable by Western immunoblotting, all have been fully characterized by metabolic labeling and immunoprecipitation (unpublished results). All three antibodies recognize epitopes found on the protein cores of cell surface glycoptoteins. XF8 and AF20 recognize dissimilar epitopes on a 180 kDA homodimer which is also detectable in normal adrenal glands. SF25 recognizes a 130 kDa heterodimer also expressed by renal proximal tubule cells.
Cell homogenates Harvested FOCUS cells were centrifuged at 1000 × g for 10 min. The pellet was homogenized for 20 s on ice with a Polytron (Brinkman) in 50 vols. of PBS (pH 7.2) containing 0.1% NaN 3, to yield a final protein concentration of approximately 0.5 mg/ml. Filter binding assay Assays were performed in 96-well plates containing glass filters (V & P Scientific, San Diego, CA) as previously described (Wilson et al., 1988). All incubations were performed at room temperature unless otherwise specified. Briefly, FOCUS cell homogenate (50 or 100 lug of protein) was applied to each filter with gentle aspiration to trap proteins. Filters were then incubated with 250 lul of 100% calf serum (CS) for 15 min to block nonspecific protein binding sites. After filtering
off the calf serum, wells were preincubated for 15 min with 50% C S / P B S or with 100 lul of a 1/100 dilution of unlabeled blocking mAb in 50% CS/PBS. To each well, 105 cpm of 125I-labeled mAb diluted in 50% C S / P B S was added and incubated for 2 h. Filters were washed five times (1.5 ml/well) with 5% C S / P B S and counted for gamma radioactivity. The data obtained from several different experiments were normalized with respect to specific binding. Specific binding was determined by subtracting the binding of radiolabeled mAb in the presence of unlabeled homologous mAb (nonspecific binding) from the unblocked binding of radiolabeled mAb (total binding).
Antigen stability assays The effect on epitope immunoreactivity of exposing the antigens to physiochemical modifications was assayed using the above filter binding assay with the following experimental manipulations. The effect of formalin, glutaraldehyde and alcohols on epitope stability was studied by incubating the filter-bound FOCUS homogenate in the various reagents for one hour. The filters were then aspirated dry and washed five times with PBS pH 7.2 (1.5 ml/well) prior to addition of blocking calf serum. To study the effect of ethanol, acetone, and xylene, filters containing bound homogenate were gently removed from 96-well plates and incubated in the organic solvent for 1 h. The solvent was evaporated off, and the filters were replaced in the wells. The filter binding assay was then performed as above. Thus the effects of ethanol were studied by two methods, which gave nearly identical results, indicating that the observed immunoreactive modifying effects of ethanol were not due to washing away of antigen bound to the filter plates. The effect of pH on antigen stability was determined by incubating filters containing bound FOCUS homogenate with various pH solutions of PBS (200 lul). The pH solutions were then aspirated and filters were washed five times (1.5 ml/well) with 5% C S / P B S pH 7.2 prior to addition of blocking calf serum. An alternative method, which gave identical results, was to preincubate the FOCUS homogenate with various pH solutions for 30 min followed by neutralization of the solu-
58
tions to pH 7 prior to binding the homogenate to the filter. Thermoinactivation studies were conducted by heating FOCUS homogenates in glass tubes at the desired temperature for 1 h, or at 1 0 0 ° C for 20 min. After heating, the homogenates were allowed to gradually cool to room temperature to allow for any reversible processes to occur. Any precipitates were gently resuspended. The cell homogenates were then applied to filters and assayed as described above.
Kinetic studies T h e kinetics of t h e r m a l d e n a t u r a t i o n were studied by a d d i n g F O C U S h o m o g e n a t e to glass tubes p r e h e a t e d in a 75 ° C r o t a t i n g water bath. A t t i m ed intervals, the h o m o g e n a t e s were r e m o v e d f r o m the w at er b a t h an d further t h e r m o i n a c t i v a tion was q u e n c h e d with 1 ml of ice-cold PBS buffer. H o m o g e n a t e s were then l o a d e d o n t o filters for assay. T o study the effect of f o r m a l i n fixation with time, cell h o m o g e n a t e s were first b o u n d to filters
XF8
5°°°IAF5
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C
SF25
AF20
IOOC
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Fig. l. The effect on epitope immunoreactivity of transient exposure to solvents and fixatives. For four epitopes (recognized by AF5, AF20, XF8 and SF25) there is a range of intermediate solvent polarities destructive to immunoreactivity. The positive control was normal saline.
59
then incubated with 200 ffl of 10% formalin in PBS for various time intervals. Filters were washed 5 times with PBS and assayed as described above.
~
-8
F20
• Melhonol i c Elhonor 1 IsoproponoI
Lb
t
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-
15
Results
Six anti-FOCUS monoclonal antibodies (named AF5, AF20, FB50, SF25, SF90, and XF8) were selected for this study because the antigens they recognize have been previously well characterized with respect to tissue distribution, biochemical properties and some of their molecular characteristics (Takahashi et al.,, 1988, 1989a,b; Wilson et al., 1988; Ozturk et al., 1989; unpublished results). All are known to be at least partially protein in nature. Fig. 1 is a summary of three experiments on the effects of transient exposure to solvents and tissue fixatives on the immunoreactivity of these antigens. The solvents were selected to represent a broad spectrum of polarity. All epitopes were found to be stable in saline solutions. Four antigens (recognized by mAbs AF5, AF20, SF25, and XF8) were highly sensitive to treatment with 80% ethanol, a solvent of moderate polarity. Exposure to 100% ethanol, which is less polar, destroyed the XF8 and SF25 epitopes while only mildly affecting the immunoreactivities of the AF5 and AF20 epitopes. Treatment with acetone, a weakly polar solvent, reduced the immunoreactivities of only the XF8 and SF25 epitopes. Xylene, a non-polar solvent, did not have an effect on the immunoreactivities of any of the epitopes studied. Thus it appears that for four of the six antigens there is a range of solvent polarities that reduces immunoreactivity, whereas the other two antigens (recognized by FB50 and SF90) appear stable following exposure to all solvent polarities tested. To more closely examine the effect of polarity on epitope reactivity, ethanol solutions of varying concentrations were studied. As shown in Fig. 2, the immunoreactivity of the XF8 epitope drops quickly as ethanol concentration is raised (or as solvent polarity is lowered) and remains depressed at 100% ethanol. The AF5 and AF20 epitopes are also unstable in ethanol solutions. However in contrast to XFS, these two epitopes are more stable at the extremes of polarity. They are not
ic, I£
40
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:5
40
80
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80
40
80
PERCENT ALCOHOL
Fig. 2. The effect on epitope immunoreactivity of transient exposure to alcohols diluted with water to achieve gradations of solvent polarities. The alcohols in order of decreasing polarity are methanol, ethanol, and isopropanol. The immunoreactivity curves for the antigens recognized by AF5 and AF20 shift according to alcohol polarity, indicating that a set range of solvent polarities are destructive to epitope immunoreactivity.
inactivated until solvent polarities lower than those which destroy the XF8 epitope are reached and they are only partially destroyed at the even lower polarity of 100% ethanol, a polarity at which XF8 is still unstable. The hypothesis that there is a window of intermediate solvent polarity within which given antigens are unstable but outside of which they are stable is further supported with the finding that an alcohol of lower polarity than ethanol (isopropanol) gave a curve shifted to the left of that for ethanol whereas an alcohol of
~l
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Fig. 3. The kinetics of epitope inactivation on heating to 75 ° C (A) and fixing with 3.7% formaldehyde (B). Thermodestruction of the epitope recognized by XF8 is very rapid and exhibits first order kinetics. The kinetics of inactivation by formaldehyde crosslinking is of high order and is concentration dependent (data not shown).
60
higher polarity (methanol) gave a curve shifted to the right of that for ethanol. It appears from the above studies, that the six epitopes can be divided into three groups based upon their stability in solvents of varying polarity: the FB50 and SF90 defined epitopes show stability in all polarities tested; the AF20 and AF5 epitopes show instability in a narrow range of polarities corresponding to solutions of 40-90% ethanol; and the XF8 and SF25 epitopes show a broad range of instability starting at high polarity (30% ethanol) and continuing past the low polarity of 100% acetone. Fig. 1 shows the effects of cross-linking fixatives on immunoreactivity. The epitopes recognized by five of the six antibodies studied (AF5, AF20, XF8, SF25, and FB50) were sensitive to 2% glutaraldehyde such that virtually all of the binding activity was destroyed. In contrast, formaldehyde substantially altered the immunoreactivity of only one epitope (XF8), and mildly lowered the reactivity of two others (AF20 and SF25). Thus we found glutaraldehyde to be the more immunodestructive of the two crosslinking agents; affecting even the remarkably stable epitope recognized by FB50. The fixative-induced decrease in immunoreactivity varied with concentration (data not shown) and duration of fixation. Fig. 3B demonstrates the time course of inactivation for the epitope recognized by XF8 using 3.7% formaldehyde. The AF20 and SF25 defined epitopes demonstrated similar kinetics, but with slightly longer half lives (data not shown). Fig. 4 illustrates results of four experiments on the effects of temporarily heating the antigens. The epitopes recognized by AF5 and SF25 are abruptly thermoinactivated between 4 2 ° C and 55°C whereas the XF8 and AF20 epitopes are thermoinactivated between 55 ° C and 65°C. The FB50 and SF90 epitopes were quite stable, even if boiled for 20 min. The destruction of thermal-sensitive epitopes occurs very rapidly. Fig. 3A illustrates two experiments on the kinetics of thermoinactivation for the XF8 epitope. Similar results were obtained for the antigens recognized by AF5 and AF20 (data not shown). The thermoinactivation of our epitopes occurred with half-lives of less than 30 s at 7 5 ° C (Fig. 3A); findings consistent with non-co-
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55
© 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100160A
JIM 05912
Stability of monoclonal antibody-defined epitopes * Byron E. Wilson, Susan Sun, M e h m e t O z t u r k a n d Jack R. W a n d s Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, Charlestown, MA 02129, U.S.A., and Department of Medicine, Harvard Medical School, Boston, MA 02114, U.S.A. (Received 28 December 1990, accepted 15 January 1991)
Epitope instability can limit the applications of monoclonal antibody (mAb) technology in laboratory and clinical research. We exposed a group of representative antigens on human hepatocellular carcinoma (HCC) cells to physiochemical insults to study epitope stability as measured by mAb immunoreactivity. Each epitope was found to have a unique pattern of instability which serves to biophysically characterize the antigen and de'fines the conditions to which the antigen can be exposed during laboratory and clinical investigations. Individual antigens were found to be unstable within a surprisingly well defined window of solvent polarities while being stable on either side of that window. Several antigens were observed to be unstable when exposed to transient changes in pH. When a critical temperature between 42 ° C and 65 °C was achieved, epitopes which were thermosensitive underwent a sudden loss in immunoreactivity. This critical temperature was found to be pH dependent. The effects of polarity, pH, and temperature on epitope stability are consistent with changes in protein structural conformation. In addition, we found that certain fixatives cause a time and concentration dependent loss of epitope immunoreactivity. This study provides a rapid and easy determination of monoclonal antibody-defined epitope stability; the results of which serve to guide further studies on the antigen and to characterize the antigen on the basis of its unique physiochemical stability. Key words." Cell surface antigen; Epitope; Monoclonal antibody
Introduction
The development of monoclonal antibody (mAb) technology has led to a new generation of
Correspondence to: J.R. Wands, Molecular Hepatology Laboratory, M G H Cancer Center, 7th Floor, M G H East, 149 13th Street, Charlestown, MA 02129, U.S.A. * This work was supported in part by Grants CA-3571, AA02666, AA-01862 and HD-20469 from the National Institutes of Health and a grant from Association pour la Recherche sur le Cancer, Villejuif, France. J.R.W. is the recipient of a Research Scientist Award AA-00048.
research and clinical reagents. Morloclonal antibodies are now instrumental in antigen identification, characterization, and purification; in cloning of translated genetic products; and in immunohistology for investigative and diagnostic purposes (Gatter et al., 1984; Tainer et al., 1984; DeLellis et al., 1987; Takahashi et al., 1988, 1989a, b; Ozturk et al., 1989). In addition, monoclonal antibodies are becoming increasing useful as potential in vivo immunotargeting and therapeutic reagents. (Vitetta et al., 1987; Takahashi et al., 1988, 1989a, b). cad The advantage of monoclonal over polyclonal antibodies is derived from the exquisite specificity
56 afforded by single epitope recognition. However, the potential applications of a given mAb can be limited if the epitope recognized by that antibody is unstable. For example, immunohistological techniques often require extensive processing of tissue specimens for preservation of morphology; Western immunoblotting requires antigen solubilization and often denaturation; protein purification may involve acidic and alkaline conditions, use of polar and nonpolar solvents, or thermal precipitation. Under such non-physiological conditions, a given epitope's immunoreactivity may be destroyed or substantially altered. Polyclonal antibodies often recognize a spectrum of many different epitopes such that physiochemical modifications destroy only a subset of epitopes, resulting in a lowered, but generally not completely lost, total immunoreactivity (Vita et al., 1985; DeLellis et al., 1987). In contrast, as a single epitope system, the immunoreactivity of an antigen to a monoclonal antibody may be completely lost following a physiochemical insult (Hancock et al., 1982; Garter et al., 1984; Oda et al., 1984; Ciocca et al., 1986; DeLellis et al., 1987; Pollard et al., 1987). Determining the stability of a given epitope provides a partial characterization of the antigen. For example, changes in immunoreactivity following physiochemical insult have been used to differentiate between (and define the structural basis for) rheumatoid factor crossidiotypes (Agnello et al., 1986); to detect subtle differences in type IV collagen between tissues (Linsenmayer et al., 1984); to study the conformational substructure of thermolysin (Vita et al., 1985); and to study the relationship between structure and function for IgE domains containing the mast cell receptor binding site (Dorrington et al., 1973; Hastings et al., 1988). On a more practical level, determining the stability of a given epitope delineates the conditions necessary for use of the monoclonal antibody as an investigative or clinical tool (Hancock et al., 1982; Oda et al., 1984; Pollard et al., 1987). We have developed methods by which one can readily determine the stability of epitopes to various physiochemical conditions often encountered in laboratory and clinical research, and have applied these methods to a panel of well defined tumor-associated antigens.
Materials and methods
Cell culture The hepatocellular carcinoma cell line FOCUS developed by us (Lun et al., 1984) was maintained in Eagle's minimum essential medium (M.A. Bioproducts, Walkersville, MD) supplemented with 10 /~M of nonessential amino acids and 10% fetal bovine serum inactivated at 56°C. Immunizing cells were grown from the second passage of the FOCUS cell line and were harvested from monolayer culture with EDTA/Versene in the absence of trypsin, then washed with PBS. Monoclonal antibody production 6-week-old B A L B / c female mice were first immunized by intraperitoneal injection of 4.0 x 10 6 FOCUS cells emulsified in 50% (v/v) complete Freund's adjuvant. Secondary immunizations were performed 6-10 weeks later by intravenous injection of 4.0 × 106 intact cells in 0.2 ml of PBS. 3 days later, mouse splenocytes were fused with either SP20, NS1, or X63 mouse myeloma cell lines with 30% (w/v) polyethylene glycol. Resulting hybridomas were maintained and selected as described (Laemmli et al., 1970). Screening for mAbs was performed by the indirect binding assay as described (Wilson et al., 1988). Purification and iodination of mAbs were performed as described (Takahashi et al., 1989; Ey et al., 1990). Properties of human tumor associated antigens We have selected six anti-FOCUS monoclonal antibodies, named AF5, AF20, FB50, SF25, SF90 and XF8, for use in this study. All these mAbs recognize antigens which share the common feature of having a high level of expression in hepatocellular carcinoma (HCC) with little or no expression in adjacent uninvolved liver (Wilson et al., 1988; unpublished results). Three antibodies (AF5, FB50, and SF90) recognize proteins identifiable by Western immunoblotting. Furthermore these mAbs are known to recognize epitopes contained within the primary structure of the protein. The antigen recognized by AF5 is a 36 kDa phosphoprotein, which in addition to HCC tissue, has been found on all transformed cell lines tested (Frohlich et al., 1990). The
57 mAb FB50 recognizes two families of 50 kDa and 100 kDa proteins (data not shown). SF90 recognizes a 50 kDa protein distinct from that recognized by FB50 (Ozturk et al., 1989). In addition to high HCC expression, the FB50 and SF90 antigens are highly expressed in normal adrenal glands but are expressed at considerably lower levels in other normal human tissues. The SF90 antigen has been present on all malignant human cells thus far tested. AF20, SF25, and XF8 recognize antigens highly expressed in adenocarcinomas of the colon, lung and liver and most other malignant tumors, with little if any expression on normal human tissues (Takahashi et al., 1988, 1989a). None of these three antigens are demonstrable by Western immunoblotting. All three antigens have been successful in imaging tumors in nude mouse models (Takahashi et al., 1988, 1989a, b). Although none of these antigens are demonstrable by Western immunoblotting, all have been fully characterized by metabolic labeling and immunoprecipitation (unpublished results). All three antibodies recognize epitopes found on the protein cores of cell surface glycoptoteins. XF8 and AF20 recognize dissimilar epitopes on a 180 kDA homodimer which is also detectable in normal adrenal glands. SF25 recognizes a 130 kDa heterodimer also expressed by renal proximal tubule cells.
Cell homogenates Harvested FOCUS cells were centrifuged at 1000 × g for 10 min. The pellet was homogenized for 20 s on ice with a Polytron (Brinkman) in 50 vols. of PBS (pH 7.2) containing 0.1% NaN 3, to yield a final protein concentration of approximately 0.5 mg/ml. Filter binding assay Assays were performed in 96-well plates containing glass filters (V & P Scientific, San Diego, CA) as previously described (Wilson et al., 1988). All incubations were performed at room temperature unless otherwise specified. Briefly, FOCUS cell homogenate (50 or 100 lug of protein) was applied to each filter with gentle aspiration to trap proteins. Filters were then incubated with 250 lul of 100% calf serum (CS) for 15 min to block nonspecific protein binding sites. After filtering
off the calf serum, wells were preincubated for 15 min with 50% C S / P B S or with 100 lul of a 1/100 dilution of unlabeled blocking mAb in 50% CS/PBS. To each well, 105 cpm of 125I-labeled mAb diluted in 50% C S / P B S was added and incubated for 2 h. Filters were washed five times (1.5 ml/well) with 5% C S / P B S and counted for gamma radioactivity. The data obtained from several different experiments were normalized with respect to specific binding. Specific binding was determined by subtracting the binding of radiolabeled mAb in the presence of unlabeled homologous mAb (nonspecific binding) from the unblocked binding of radiolabeled mAb (total binding).
Antigen stability assays The effect on epitope immunoreactivity of exposing the antigens to physiochemical modifications was assayed using the above filter binding assay with the following experimental manipulations. The effect of formalin, glutaraldehyde and alcohols on epitope stability was studied by incubating the filter-bound FOCUS homogenate in the various reagents for one hour. The filters were then aspirated dry and washed five times with PBS pH 7.2 (1.5 ml/well) prior to addition of blocking calf serum. To study the effect of ethanol, acetone, and xylene, filters containing bound homogenate were gently removed from 96-well plates and incubated in the organic solvent for 1 h. The solvent was evaporated off, and the filters were replaced in the wells. The filter binding assay was then performed as above. Thus the effects of ethanol were studied by two methods, which gave nearly identical results, indicating that the observed immunoreactive modifying effects of ethanol were not due to washing away of antigen bound to the filter plates. The effect of pH on antigen stability was determined by incubating filters containing bound FOCUS homogenate with various pH solutions of PBS (200 lul). The pH solutions were then aspirated and filters were washed five times (1.5 ml/well) with 5% C S / P B S pH 7.2 prior to addition of blocking calf serum. An alternative method, which gave identical results, was to preincubate the FOCUS homogenate with various pH solutions for 30 min followed by neutralization of the solu-
58
tions to pH 7 prior to binding the homogenate to the filter. Thermoinactivation studies were conducted by heating FOCUS homogenates in glass tubes at the desired temperature for 1 h, or at 1 0 0 ° C for 20 min. After heating, the homogenates were allowed to gradually cool to room temperature to allow for any reversible processes to occur. Any precipitates were gently resuspended. The cell homogenates were then applied to filters and assayed as described above.
Kinetic studies T h e kinetics of t h e r m a l d e n a t u r a t i o n were studied by a d d i n g F O C U S h o m o g e n a t e to glass tubes p r e h e a t e d in a 75 ° C r o t a t i n g water bath. A t t i m ed intervals, the h o m o g e n a t e s were r e m o v e d f r o m the w at er b a t h an d further t h e r m o i n a c t i v a tion was q u e n c h e d with 1 ml of ice-cold PBS buffer. H o m o g e n a t e s were then l o a d e d o n t o filters for assay. T o study the effect of f o r m a l i n fixation with time, cell h o m o g e n a t e s were first b o u n d to filters
XF8
5°°°IAF5
t00C 80C 60C
L
20O0
40C
20C
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C
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AF20
IOOC
3000
80C 2000
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Fig. l. The effect on epitope immunoreactivity of transient exposure to solvents and fixatives. For four epitopes (recognized by AF5, AF20, XF8 and SF25) there is a range of intermediate solvent polarities destructive to immunoreactivity. The positive control was normal saline.
59
then incubated with 200 ffl of 10% formalin in PBS for various time intervals. Filters were washed 5 times with PBS and assayed as described above.
~
-8
F20
• Melhonol i c Elhonor 1 IsoproponoI
Lb
t
20
-
15
Results
Six anti-FOCUS monoclonal antibodies (named AF5, AF20, FB50, SF25, SF90, and XF8) were selected for this study because the antigens they recognize have been previously well characterized with respect to tissue distribution, biochemical properties and some of their molecular characteristics (Takahashi et al.,, 1988, 1989a,b; Wilson et al., 1988; Ozturk et al., 1989; unpublished results). All are known to be at least partially protein in nature. Fig. 1 is a summary of three experiments on the effects of transient exposure to solvents and tissue fixatives on the immunoreactivity of these antigens. The solvents were selected to represent a broad spectrum of polarity. All epitopes were found to be stable in saline solutions. Four antigens (recognized by mAbs AF5, AF20, SF25, and XF8) were highly sensitive to treatment with 80% ethanol, a solvent of moderate polarity. Exposure to 100% ethanol, which is less polar, destroyed the XF8 and SF25 epitopes while only mildly affecting the immunoreactivities of the AF5 and AF20 epitopes. Treatment with acetone, a weakly polar solvent, reduced the immunoreactivities of only the XF8 and SF25 epitopes. Xylene, a non-polar solvent, did not have an effect on the immunoreactivities of any of the epitopes studied. Thus it appears that for four of the six antigens there is a range of solvent polarities that reduces immunoreactivity, whereas the other two antigens (recognized by FB50 and SF90) appear stable following exposure to all solvent polarities tested. To more closely examine the effect of polarity on epitope reactivity, ethanol solutions of varying concentrations were studied. As shown in Fig. 2, the immunoreactivity of the XF8 epitope drops quickly as ethanol concentration is raised (or as solvent polarity is lowered) and remains depressed at 100% ethanol. The AF5 and AF20 epitopes are also unstable in ethanol solutions. However in contrast to XFS, these two epitopes are more stable at the extremes of polarity. They are not
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Fig. 2. The effect on epitope immunoreactivity of transient exposure to alcohols diluted with water to achieve gradations of solvent polarities. The alcohols in order of decreasing polarity are methanol, ethanol, and isopropanol. The immunoreactivity curves for the antigens recognized by AF5 and AF20 shift according to alcohol polarity, indicating that a set range of solvent polarities are destructive to epitope immunoreactivity.
inactivated until solvent polarities lower than those which destroy the XF8 epitope are reached and they are only partially destroyed at the even lower polarity of 100% ethanol, a polarity at which XF8 is still unstable. The hypothesis that there is a window of intermediate solvent polarity within which given antigens are unstable but outside of which they are stable is further supported with the finding that an alcohol of lower polarity than ethanol (isopropanol) gave a curve shifted to the left of that for ethanol whereas an alcohol of
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Fig. 3. The kinetics of epitope inactivation on heating to 75 ° C (A) and fixing with 3.7% formaldehyde (B). Thermodestruction of the epitope recognized by XF8 is very rapid and exhibits first order kinetics. The kinetics of inactivation by formaldehyde crosslinking is of high order and is concentration dependent (data not shown).
60
higher polarity (methanol) gave a curve shifted to the right of that for ethanol. It appears from the above studies, that the six epitopes can be divided into three groups based upon their stability in solvents of varying polarity: the FB50 and SF90 defined epitopes show stability in all polarities tested; the AF20 and AF5 epitopes show instability in a narrow range of polarities corresponding to solutions of 40-90% ethanol; and the XF8 and SF25 epitopes show a broad range of instability starting at high polarity (30% ethanol) and continuing past the low polarity of 100% acetone. Fig. 1 shows the effects of cross-linking fixatives on immunoreactivity. The epitopes recognized by five of the six antibodies studied (AF5, AF20, XF8, SF25, and FB50) were sensitive to 2% glutaraldehyde such that virtually all of the binding activity was destroyed. In contrast, formaldehyde substantially altered the immunoreactivity of only one epitope (XF8), and mildly lowered the reactivity of two others (AF20 and SF25). Thus we found glutaraldehyde to be the more immunodestructive of the two crosslinking agents; affecting even the remarkably stable epitope recognized by FB50. The fixative-induced decrease in immunoreactivity varied with concentration (data not shown) and duration of fixation. Fig. 3B demonstrates the time course of inactivation for the epitope recognized by XF8 using 3.7% formaldehyde. The AF20 and SF25 defined epitopes demonstrated similar kinetics, but with slightly longer half lives (data not shown). Fig. 4 illustrates results of four experiments on the effects of temporarily heating the antigens. The epitopes recognized by AF5 and SF25 are abruptly thermoinactivated between 4 2 ° C and 55°C whereas the XF8 and AF20 epitopes are thermoinactivated between 55 ° C and 65°C. The FB50 and SF90 epitopes were quite stable, even if boiled for 20 min. The destruction of thermal-sensitive epitopes occurs very rapidly. Fig. 3A illustrates two experiments on the kinetics of thermoinactivation for the XF8 epitope. Similar results were obtained for the antigens recognized by AF5 and AF20 (data not shown). The thermoinactivation of our epitopes occurred with half-lives of less than 30 s at 7 5 ° C (Fig. 3A); findings consistent with non-co-
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