Monoclonal Antibody against Diphtheria Toxin EFFECT ON TOXIN BINDING AND ENTRY INTO CELLS*

A number of monoclonal antibodies against diphthe- ria toxin were isolated. Some of their properties were determined. Antibody 2 reacts with the region of be- tween 30 and 46 kDa from the N H 2 terminus of toxin. Antibody 7 reacts with the COOH-terminal 17-kDa re- gion of toxin. These two antibodies show sharp con-trasts in their effects on toxin action in cultured cells. When antibody 2 or 7 and toxin were mixed, incubated at 37 “C, and then added to sensitive Vero cells, antibody 7 blocked toxin action, but antibody 2 did not. When antibody 2 or 7 was added to cells to which toxin had been prebound at 4 “C, and the cells were then shifted to 37 “C, antibody 7 did not block toxin action, but antibody 2 inhibited intoxication. Antibody 7 blocked binding of 1261-toxin to cells and did not block degradation of toxin associated with cells. Antibody 2 did not block binding of 1261-toxin to cells, and was able to bind to cells in the presence of toxin. The results obtained from the effect of antibody 2 on degradation of i251-toxin associated with cells resemble those seen with amines, which block toxin action but do not inhibit binding of toxin to cells. These facts show that antibody 2 does not block binding of toxin to cell surfaces, but blocks the entry of toxin into the cytosol at a step after binding of toxin to the receptor. Antibodies 14 and 15 react with fragment A of diphtheria toxin, but have no effect on any activity of toxin. The other monoclonal antibodies have effects on toxin binding and entry in- termediate between those

A number of monoclonal antibodies against diphtheria toxin were isolated. Some of their properties were determined. Antibody 2 reacts with the region of between 30 and 46 kDa from the N H 2 terminus of toxin. Antibody 7 reacts with the COOH-terminal 17-kDa region of toxin. These two antibodies show sharp contrasts in their effects on toxin action in cultured cells. When antibody 2 or 7 and toxin were mixed, incubated at 37 "C, and then added to sensitive Vero cells, antibody 7 blocked toxin action, but antibody 2 did not. When antibody 2 or 7 was added to cells to which toxin had been prebound at 4 "C, and the cells were then shifted to 37 "C, antibody 7 did not block toxin action, but antibody 2 inhibited intoxication. Antibody 7 blocked binding of 1261-toxin to cells and did not block degradation of toxin associated with cells. Antibody 2 did not block binding of 1261-toxin to cells, and was able to bind to cells in the presence of toxin. The results obtained from the effect of antibody 2 on degradation of i251-toxin associated with cells resemble those seen with amines, which block toxin action but do not inhibit binding of toxin to cells. These facts show that antibody 2 does not block binding of toxin to cell surfaces, but blocks the entry of toxin into the cytosol at a step after binding of toxin to the receptor. Antibodies 14 and 15 react with fragment A of diphtheria toxin, but have no effect on any activity of toxin. The other monoclonal antibodies have effects on toxin binding and entry intermediate between those of 2 and 7.
After diphtheria toxin (MI = 62,000) binds to receptors on the surface of toxin-sensitive cells, at least its NH2-terminal fragment A (Mr = 21,150 (1)) enters the cytoplasm, where it inactivates elongation factor 2 (2-4) by ADP-ribosylation (5) and thus blocks protein synthesis. Only fragment A need reach the cytoplasm; when fragment A alone is introduced directly into the cytoplasm the cells are killed (6,7).
The entry of the toxin into cells has recently been studied extensively, but a number of questions remain unanswered. There are two approaches to the problem. One is to study the cellular functions and environment necessary for entry of the toxin. Receptors for diphtheria toxin have been partially characterized, although they have not yet been isolated in intact form (8,9). CRM45, a prematurely terminated mutant toxin protein (lo), and toxin itself have been shown to form ion-permeable channels in artificial membranes at low pH (approximately 5), but not at neutral pH (11,12), and recent * The costa of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. work suggests that diphtheria toxin crosses the cell membrane only after exposure to low pH in an intracellular vesicle (13,14).
The second approach is to determine what structural features of toxin are required for entry into cells. Several lines of investigation have provided information on the relationship between the structure of diphtheria toxin fragment B and its role in toxin entry. The COOH-terminal region of the toxin is required for binding to cell surface receptors, as CRM45, which lacks the COOH-terminal 17-kDa region of toxin, cannot bind to cells (15). There are hydrophobic regions located in the middle portion of the toxin sequence (Le. the portion of fragment B that remains in CRM45) (16,17). The importance of the hydrophobic regions for entry into cells is suggested by comparison of the cytotoxicities of hybrid proteins constructed from Wistaria floribunda lectin and ricin A subunit, which contains a hydrophobic region, and wistaria lectin and diphtheria toxin fragment A, which does not. The hybrid protein containing the ricin A subunit is 100-200 times more toxic than the hybrid protein containing diphtheria toxin fragment A (18). Similar results have been obtained with hybrids of epidermal growth factor and ricin A subunit or diphtheria fragment A (19). Diphtheria toxin has also been shown to contain apolyanion binding site, the P-site (20,21). The P-site is located within the strongly cationic M, = 8000 COOH-terminal cyanogen bromide fragment of the toxin. In most toxin preparations, some of the toxin molecules have a dinucleotide bound to the P-site. Although binding of nucleotides to the P-site may inhibit the initial interaction of toxin with its receptor, the role of the P-site in the entry process remains unclear.
To obtain further information on structure-function relationships of diphtheria toxin, we isolated monoclonal antibodies against toxin and determined which functional domains of the toxin they recognized. We describe here isolation of 14 monoclonal antibodies against diphtheria toxin. These include one that reacts with the region of between 30 and 45 kDa from the NH2 terminus of toxin and does not block binding of toxin to the cell surface, but does prevent the entry of toxin into the cell. Another antibody, which binds to the COOH-terminal 17-kDa region, blocks binding of toxin to cells.
Radioiodination of Proteins-Toxin, CRMs, and IgG were labeled with Na"'1 using chloramine-T (23). The labeled proteins were fdtered through nitrocellulose fdters in the presence of 1 mg/ml of bovine serum albumin to reduce nonspecific binding.
Cells a n d Cell Culture-Vero cells and L-cells were cultured in MEM' supplemented with 10% calf serum. SP2/0-Ag 14 myeloma cells were cultured in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum.
Isolation of Monoclonal Antibody-BALB/c mice (8 weeks old) were injected intraperitoneally with 0.1 mg of formalinized diphtheria toxin (24) in complete Freund's adjuvant. Two weeks later, 0.1 mg of the toxoid in incomplete Freund's adjuvant was injected intraperitoneally, and after 2 more weeks, 0.1 mg of toxoid was injected intraperitoneally. Four days later, spleen cells (2 X IO") from the immunized mice were fused with SP2/0 cells (2 X IO') using 50% (w/v) polyethylene glycol 4000 and 15% (v/v) dimethyl sulfoxide (25). After fusion, the cells were seeded in 96-well, tissue culture plates and cultured in HAT (1 X 10" M hypoxanthine, 4 X lo-' M aminopterin, showed good growth after 10 days in selective medium. Cells were grown for a total of 14 days in HAT medium and gradually adapted to Dulbecco's modified Eagle's medium with 15% fetal calf serum by passage for 7 days in medium lacking aminopterin but containing hypoxanthine and thymidine. Hybridoma culture supernatants (100 pl) were incubated with 12'1labeled diphtheria toxin (20,000 cpm, 0.5 ng) in 0.1 M borate, pH 8.6, containing 0.2% bovine serum albumin. After 3 h, 100 pl of 1% bovine y-globulin in 0.1 M borate, pH 8.6, was added as carrier IgG, followed by 1 ml of 20% polyethylene glycol 6000 in the same buffer (26). After thorough mixing, the samples were centrifuged a t 3000 rpm for 15 min and the supernatants were removed. The radioactivity remaining in the tube was counted in a y counter. Cells secreting the desired antibodies were cloned by the limiting dilution method using macrophages as feeder cells. The cloning procedure was repeated a second time.
The cloned hybridoma cells that produced antibody against diphtheria toxin were cultured in bulk and 2 X 10" cells were injected into the peritoneum of BALB/c mice that had been injected intraperitoneally with 0.5 rnl of pristane 2 weeks previously. After 10-12 days, ascites containing antibody was obtained from the mice. Monoclonal antibodies were purified using DE52 and diphtheria toxin-conjugated Sepharose 4B. Immunodiffusion tests using antibodies specific for IgG and IgM showed that antibodies 1, 2,5, 7, and 15 were IgG.
Radioimmunoprecipitation a n d SDS-Polyacrylamide Gel Electrophoresis-Samples of each monoclonal antibody (10 pg) were added separately to "'I-labeled toxin or toxin-related proteins (1 pg, -500,000 cpm) in a total volume of 400 pl. The mixture was incubated at 4 "C for 24 h, and then 10 1-11 of rabbit anti-mouse IgG serum was added and the incubation was continued for another 24 h. Protein A-Sepharose 4B (30 pl) was added and the mixtures were incubated 24 h at 4 "C. The Sepharose was collected by centrifugation and washed three times with 0.9% NaCl containing 10 mM Tris, pH 8.0, and then bound protein was eluted with 50 pl of sample buffer containing 0.06 M Tris-HCI, pH 6.8, 3% SDS, and 5% glycerol. The eluates were electrophoresed in 11% polyacrylamide-SDS gels using a minor modification of the method of Laemmli (27). The gels were dried and exposed to Kodak X-Omat S film for 2 days.
Inhibition of Binding of '2'Z1-labeled Monoclonal Antibodies to Diphtheria Toxin-Sepharose by Other Monoclonal Antitoxins-Diphtheria toxin-Sepharose 4B (bound toxin, 40 ng; solution volume, 100 PI) was added to each antibody (40 pg in 200 p1 of Tris-buffered saline, pH 8.3) and the mixture was rotated a t 4 OC for 24 h. "' Ilabeled antibody (400 ng, -4 X 1 0 cpm) in 100 pl of borate-buffered saline, pH 8.0, was then added and the mixture was incubated an additional 24 h at 4 "C. The Sepharose was washed three times with borate-buffered saline and counted in a y counter.
Association Constants of Monoclonal Antibodies for Toxin or CRM45-The association constants of each antibody for toxin and CRM45 were determined by a competitive binding assay (28) using 1.4-685 ng of "'I-toxin (1.32 X 10" cpm/pg) or 1.0-500 ng of 12'1-I The abbreviations used are: MEM, Eagle's minimal essential medium; SDS, sodium dodecyl sulphate; PBS, phosphate-buffered saline; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. CRM45 (6.4 X lo' cpm/pg), unlabeled 0.2 pg of toxin, or 0.2 pg of CRM45, and each monoclonal antibody conjugated to Sepharose. Labeled and unlabeled toxin or CRM45 and each antibody were mixed and rotated for 48 h a t 4 "C. Then the Sepharose was collected, washed with 10 mM Tris (pH 7.5) containing 0.15 M NaCl and 2mg/ ml of bovine serum albumin and counted in a y counter.
Assay of the Rate of Protein Synthesis in Cells Treated with Diphtheria Toxin a n d Monoclonal Antibody-The assay method was described previously (29). Vero cells were seeded into 24-well tissue culture plates and incubated for 24 h in MEM with 10% calf serum. T h e cells were washed with Dulbecco's PBS, and then 0.5 ml of low leucine MEM (leucine concentration, 1:lO) containing 5% calf serum and 20 mM HEPES buffer, pH 7.2, was added to each well. After treatment with diphtheria toxin and antibody (details given in the legend to Fig. 2) the cells were incubated 1-3 h at 37 "C. They were then labeled with 2 pCi/ml of ['Hlleucine for 60 min. The cells were washed with PBS and dissolved in 1 rnl of 0.1 N NaOH. An equal volume of 20% trichloroacetic acid was added to each sample. The precipitates were collected and washed with 5% trichloroacetic acid on a glass fiber filter, dried, and counted in a liquid scintillation system. The rate of protein synthesis in each culture was expressed as a percentage of the value obtained in cultures without diphtheria toxin. Control values in various experiments were 20,000 to 40,000 cpm.
Effect of Monoclonal Antibodies on Association of '*'Z-Toxin with Cells-The association of iodinated diphtheria toxin with cells was examined using the method of Middlebrook et al. (30). Vero cells were seeded in tissue culture dishes and cultured 2 days. The medium was replaced with MEM containing 10% calf serum and 25 mM HEPES buffer, pH 7.2. "'I-labeled diphtheria toxin and monoclonal antibody, preincubated 30 min at 37 "C and at 4 "C overnight, were then added to the dishes. The cells were incubated for the times indicated in Fig. 3, washed four times with Dulbecco's phosphatebuffered saline containing 0.2 r " CaCL, and dissolved in 0.1 N NaOH. The samples were transferred to test tubes and counted in a y counter. The values shown are specific counts, determined by subtracting the counts obtained with cells incubated with '*'I-toxin and a 100-fold excess of unlabeled toxin from the counts obtained with '*'I-toxin alone or with '''I-toxin and antibody. In these experiments, binding of '*'I-toxin in the presence of a 100-fold excess of unlabeled toxin was 10% or less of the total binding. This suggests that antibody 7 reacts with a determinant located within the COOH-terminal 17-kDa region of diphtheria toxin, 14 and 15 react with determinants on fragment A, and the remaining antibodies recognize determinants in the region 30 to 45 kDa from the NH2 terminus. Competition between Monoclonal Antibodies for Binding to Diphtheria Toxin Linked to Sepharose-Monoclonal antibodies 1, 2, 5, and 7 were labeled with lz5I. Binding of the labeled antibodies to diphtheria toxin-Sepharose and to diphtheria toxin-Sepharose preincubated with a 100-fold greater amount of unlabeled antibody was measured as described under "Experimental Procedures." The unlabeled antibody in each case completely inhibits binding of the homologous lz5Ilabeled antibody, while pairs of unlike antibodies show essentially symmetrical inhibition of varying degree ( Table I).

Binding
Inhibition of Toxin Action by Monoclonal Antibodies-Five antibodies were selected for use in the following experiments. The association constants of each antibody for diphtheria toxin and CRM45 are shown in Table 11. Antibody 7 does not bind to CRM45. Although 15 binds well to fragment A, it has a low affiity for intact toxin. The effect of these antibodies on the inhibition of protein synthesis in cells exposed to diphtheria toxin was studied in two ways. In one set of experiments, toxin and antibody were preincubated and then added to cultured cells, and the rate of protein synthesis was determined at several times after the addition of the mixture. In the second set of experiments, toxin was prebound to cells at 4 "C. The cells were washed, and then antibody was added. The cells were incubated 2 h at 4 "C with the antibody and then warmed to 37 "C, and the rate of protein synthesis was measured at various times after warming the cells.
As seen in Column A of Table 11, toxin incubated with antibody 7 or 5 is almost completely neutralized. Antibody 2 has little effect on the action of toxin under these conditions. However, when the antibodies were added to cells to which toxin had been prebound, antibody 2 prevented the inhibition of protein synthesis by diphtheria toxin. Under these conditions, antibody 7 was not able to block the effect of toxin. As antibodies 2 and 7 had very different activities in the two assays, we examined further the effects of these antibodies on the kinetics of inhibition of protein synthesis in cells exposed to toxin. These studies confiied that 2 and 7 have distinctly different effects (Fig. 2).
These results suggested that antibody 7 blocks the binding of toxin to receptors on the cell surface, and that 2 blocks the entry of toxin into the cytosol at a step after binding of toxin to the receptor. Additional evidence for these interpretations was obtained in the experiments described below.
Effect of Monoclonal Antibodies on the Binding of Toxin to Cells-Because antibodies 2 and 7 showed contrasting effects in the assays described above, we studied their effects on lZ5I-diphtheria toxin binding to cells. As shown in Fig. 3A, antibody 7 inhibits almost completely the binding of toxin to cells at high concentrations of antibody, but antibody 2 does not effectively block toxin binding, and binding equal to 75% of the control value is observed in the presence of an 80-fold excess of antibody. At a ratio of antibody to toxin of 2 5 1 , binding of toxin to cells was lowered to about 40% of the control value by antibody 7, while antibody 2 had no effect.
The kinetics of '251-toxin binding to cells was also studied

TABLE I1
Effect of various monoclonal antibodies on inhibition ofprotein synthesis by diphtheria toxin and association constant of these monoclonal antibodies with toxin and CRM45 Column A, 5 ng of diphtheria toxin and 250 or 2500 ng of antibody were mixed in 20 p1 of PBS (pH 7.2) and incubated at 37 "C for 30 min and then at 4 "C overnight. The mixture was added to Vero cells (2.5 X IO4 cells in Inhibition of protein synthesis in cells exposed to diphtheria toxin and monoclonal antibody 2 or 7. A, diphtheria toxin (5 ng) was incubated with 2.5 pg of monoclonal antibody (molar ratio of antibody to toxin, 2 0 0 3 in 20 pl at 37 "C for 30 min and then overnight at 4 "C. The mixture was added to Vero cells in 0.5 ml of medium (toxin concentration, 10 ng/ml), and the cells were incubated at 4 "C for 1 h. The cells were then washed-four times with about 1 ml of PBS Containing 0.2 m~ CaC12 in the cold (4 "C). They were then warmed to 37 "C in 1 ml of medium and incubated for the indicated times. The rate of protein synthesis was determined by pulse labeling with [3H]leucine as described under "Experimental Procedures." B, Vero cells were incubated with 40 ng/ml of diphtheria toxin for 1 h at 4 "C and then washed four times with cold PBS containing 0.2 mM CaCL to remove unbound toxin. They were then incubated for 2 h at 4 "C with 30 pg of antibody in 1 ml of medium. The cells were then warmed to 37 "C, and at various times the rate of protein synthesis was determined by pulse labeling with [3H]leucine. A and B, (A) toxin with normal mouse IgG (0) toxin with 2 antibody; (0) toxin with 7 antibody. (Fig. 3B). Antibody 7 blocked the binding of toxin as expected. Antibody 2 had no effect on the kinetics of binding of toxin at 4 "C. To determine whether antibody 2 became associated with the cell surface along with toxin, '?-labeled antibody was mixed with unlabeled toxin and incubated with cells under the same conditions as in Fig. 3B. lZ5I-Antibody 2 became associated with cells with kinetics similar to the binding of toxin (Fig. 3 0 . lZ5I-Antibody 7 did not bind to cells in the presence of toxin. In the absence of toxin, neither antibody became associated with the cells.

pl of culture medium/well) and incubated further at 37 "C. After 3 h of incubation [3H]leucine was added to each culture at a final 2 pCi/ml and incubated for 1 h. Incorporation of [3H]leucine into cells was determined as described under "Experimental Procedures." Column B, 20 ng of diphtheria toxin were added to Vero cells (2.5 X lo4 cells in 460 pl of culture medium/well) and incubated at 4 "C for 1 h. Each well was washed with PBS for
Effect of Monoclonal Antibodies on the Fate of Diphtheria Toxin Bound to Cells-At 37 "C, diphtheria toxin bound to the cell surface is internalized and degraded (30,31). To follow the effects of antibodies 2 and 7 on the fate of toxin associated with cells. '251-toxin and antibody (molar ratio of antibody to toxin of 503) were mixed and incubated at 37 "C for 30 min and then at 4 "C for 12 h. The antibody-toxin mixture was added to Vero cells at 37 "C and the amount of label associated with the cells was determined at intervals. Fig. 4A shows that in the absence of antibody the amount of label associated with the cells reached a maximum at 1 h and then decreased, as reported previously by Middlebrook et al. (30). In the presence of antibody 2, the amount of cell-associated label increased more slowly, and reached a plateau after about 4 h that was slightly higher than the peak value for cells incubated with '251-toxin in the absence of antibody. Antibody 7 blocked the association completely.
The results obtained with antibody 2 resemble those seen with amines, such as N%C1(32), chloroquine (33), and methylamine (29). These compounds block the action of toxin, although the association of toxin with cells is not blocked. Toxin accumulates in the cells to a higher level than in the absence of amines because degradation of the toxin is inhibited.
The monoclonal antibodies were also added to cells to which lZ5I-toxin had been prebound at 4 "C. Fig. 4B shows that the amount of label associated with the cells rapidly decreased in the absence of antibody. Antibody 7 had no effect on the loss of label from the cells. However, when antibody 2 was added to the cells before warming, the loss of radioactivity was slower, and the curve is similar to that for the loss of label in the presence of 10 m~ methylamine. Effect of Monoclonal Antibodies on the Intoxication of Lcells-Cell lines derived from rats and mice are resistant to the action of diphtheria toxin. Two different explanations have been given for their low sensitivity to toxin. One is that the resistant cells lack diphtheria toxin receptors (15,30), the other is that the cells have toxin-specific binding sites, but lack or have a defect in some cell component necessary for transport of the active moiety of toxin into the cytoplasm (34)(35)(36). The tissue culture LDa of toxin for mouse L-cells is about lo5 times higher than for African green monkey kidney cell lines such as Vero, BSC, and CV-1 (37,38). We examined the effects of antibodies 2 and 7 on the intoxication of L-cells.
Diphtheria toxin (20 pg) and antibody (250 pg) were incubated at 37 "C for 30 min and then at 4 "C overnight. The mixtures were added to L-cells (8 X lo4 cells/2-cm2 well) in 1 ml of medium. After 8 h incubation at 37 "C, the cells were pulselabeled with [3H]leucine (2 pCi/ml) for 1 h. The incorporation of label into trichloroacetic acid-precipitable material in cells treated with toxin and normal mouse IgG was 22% of the incorporation in cells incubated without toxin. When toxin was incubated with antibody 7, there was no effect on the degree of inhibition of protein synthesis; incorporation of label was 24% of the control. Incubation of toxin with antibody 2 reduced the effect of toxin on protein synthesis in L-cells; the incorporation of label was 86% of the control. These results are consistent with the suggestion that L-cells lack the diphtheria toxin receptor present on more sensitive cell lines.

DISCUSSION
We have prepared monoclonal antibodies against diphtheria toxin and determined the general region of the toxin molecule recognized by each of them. The relationship between diphtheria toxin structure and function was probed by examining the ability of antibodies that bind to different regions of the toxin sequence to block binding of toxin to cells, entry of prebound toxin into cells, and binding of the other antibodies to diphthena toxin linked to Sepharose. The results of these  Fig. 5. Four antibodies were "mapped" using the data from the immunoprecipitation of toxin and toxin-related proteins (Fig. 1) and the competition for binding to toxin-Sepharose (Table I) to order the antibodies. The distances marked on the abscissa of Fig. 5 are the average of the "nearest neighbor" binding competition results. The abilities of the antibodies to neutralize toxin and to prevent the entry of prebound toxin are indicated by higher rates of protein synthesis (data from Table 11). This may show relationships between the effects of an antibody on toxin action and its binding site on the toxin molecule. Antibody 7 blocks the binding of toxin to cells, and thus can neutralize toxin, but it cannot block the entry of toxin already bound to the cell. Antibody 2, which apparently recognizes a determinant in the portion of fragment B remaining in CRM45, can prevent the entry of toxin into the cytosol even after it has bound to the cell, provided the antibody is incubated with the cells before they are warmed to 37 "C. This antibody does not block binding of diphtheria toxin to the cell surface, and antibody 2 becomes associated with cells in the presence of toxin. Antibody 2 appears to block a step in toxin entry that follows binding to the cell. Antibodies 1 and 5 show effects intermediate between the contrasting antibodies 2 and 7 .
It is surprising that antibody 2 fails to protect cells from diphtheria toxin when toxin and antibody are preincubated and added to cells at 37 "C. With Vero cells, the protective effect of antibody 2 was only observed when toxin was bound to cells at low temperature, excess toxin was removed, and the cells were incubated with antibody at 4 "C before warming and measuring intoxication. This result was reproducible. A t 4 "C, antibody 2-diphtheria toxin complexes bind to the surface of Vero cells. Interaction of toxin with receptor at 37 "C, at a step after the initial binding of toxin, may compete with binding of antibody 2 to the toxin-receptor complex. Thus, some toxin can be freed from antibody and enter into the cytosol, so that a high concentration of antibody 2 and a small amount of toxin are required to ensure that a significant  Relationship between the effects of monoclonal antibodies on toxin action and recognition site of the antibodies on the toxin molecule. The binding of one antibody to diphtheria toxin-Sepharose that had been preincubated with an excess of a second antibody was taken as a measure of the "distance" between the antibody-binding sites on toxin. The least degree of inhibition was seen with the antibody pair 2 and 7, and the distance between the sites for these antibodies was set at 100. Positions of the other antibody binding sites were then determined from the data in Table  I. The distance between a pair of antibody-binding sites was calculated as the ratio of the average binding observed in competition experiments using that pair of antibodies to the binding observed when antibodies 2 and 7 were used as the labeled and unlabeled antibody.
The ratios were expressed as percentages. The effects of the monoclonal antibodies on toxin action are taken from Columns A and B of Table 11. ainst Diphtheria Toxin fraction of toxin bound to receptor is also bound to antibody (Table 11, Column B, and Fig. 2B). Under the conditions of Table 11, Column A, and Fig. 2A, some toxin that is not complexed with antibody must enter the cells. For L-cells, toxin-antibody 7 mixture was as toxic as diphtheria toxin alone. This suggests that intoxication of L-cells is not mediated by the same type receptor as is intoxication of more sensitive cell lines. Some protection was observed when toxin-antibody 2 mixture was added to L-cells at 37 "C. Thus, the failure of antibody 2 to protect Vero cells under these conditions appears due to the interaction of toxin or the toxin-antibody complex with receptor, not just to the difference in temperature.
The interpretation of the results of such studies on the effect of antibodies directed against various regions of diphtheria toxin on toxin function is complicated by the fact that antibodies are large proteins. It is unclear whether the functional region is the determinant recognized by the antibody, or a nearby region. The effect of the antibody may be indirect.
Nevertheless, we believe the simplest interpretation of the data summarized in Fig. 5 is that antibody 7 binds to toxin at or near the site recognized by the diphtheria-toxin receptor, and antibody 2 binds at or near a site involved in the entry of toxin. Thus, the present study supports the idea that fragment B of diphtheria toxin has at least two functional domains, one required for binding of toxin to cells, and another for the translocation of fragment A across the cell membrane. Antibody 2, which appears to be able to bind to toxin-receptor complexes, may be useful for isolation of such complexes from cells.