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Toxkm Vol. 29, No. 3, pp. 359-370, 1991 . Printed in GICat Britain .
PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST PSEUDEXIN JOHN L . MIDDLEBROOK Department of Toxinology, Pathology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21701-5011, U.S .A . (Received 12 lute 1990; accepted for publication 27 September 1990)
J. L. MIDDLEBROOK . Preparation and characterization of monoclonal antibodies against pseudexin. Toxicon 29, 359-370, 1991 .-Fifteen hybridoma cell lines secreting monoclonal antibodies against pseudexin were developed. The cell lines were grown as ascites tumors and the resulting antibodies were purified by Protein A amity-chromatography. Several of the antibodies exhibited extensive ELISA cross-reactions with different phospholipase A2 toxins from various snake venoms, while other of the antibodies reacted only with the pseudexins . Three of the antibodies neutralized pseudexin A and B, but none of the 10 other phospholipase A2 toxins tested . These same three antibodies inhibited the enzymatic activity of pseudexin A and B and also that of notexin. After each antibody was labeled with biotin, competition experiments were carried out to determine the binding relationships among the antibodies and the pseudexins . Competitions were frequently observed, with a low of zero to a high of eight out of the 14 possibilities. Competition experiments were also carried out with biotin-labeled rabbit IgG against the pseudexins . Some of the monoclonal antibodies had no effect on rabbit IgG binding to pseudexin, while others blocked up to 50% of the binding. INTRODUCTION
A2 (PLA2) neurotoxins are a group of structurally diverse proteins found in the venoms of crotalids, viperids and elapids. In most cases, the PLA2 toxins are the most toxic components in the venoms and probably play a major role in lethality due to snake bites. As determined in mice, PLA2 LD,0 s range from 1 yg/kg for textilotoxin (TYLER et al., 1987) to 1250 pg/kg for pseudexin A (SCHMIDT and MIDDLEBROOK, 1989). Structurally, the most complex PLA2 neurotoxin is textilotoxin with five subunits, while the simplest toxins are single chain entities, such as notexin, caudoxin or pseudexin (CHANG, 1985). Each member of this class of toxins expresses a phospholipase A2 activity, although the role enzymatic activity plays in neurotoxicity is controversial (ROSENBERG, 1986). To better understand the structural relationships among the PLA2 toxins, we have been studying their immunochemistries. We determined that extensive ELISA cross-reactions can be observed with polyclonal antisera raised to the purified neurotoxins (MIDDLEBROOK and KAISER, 1989). Based on intensities of the cross reactions, it was possible to assign each PLA2 neurotoxin to one of two serological classes: (a) elapid and viperid or (b) crotalid. The same data are also useful for determining immunological similarity of a PHOSPHOLIPASE
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given two toxins or suggest the possibility of common, neutralizing epitopes on the toxins (MIDDLEBROOK, in preparation). Previous studies showed that it was possible to obtain a monoclonal antibody that would recognize all of the crotalid neurotoxins tested KAISER and MIDDLEBROOK, 1988a) . In order to develop a similar reagent for the elapid/viperid class of PLAZ neurotoxins, monoclonal antibodies have been raised to several toxins in this group. One fusion, using pseudexin as the antigen, produced 15 clones. Properties of the monoclonal antibodies elaborated by these clones are reported here . MATERIALS AND METHODS Toxins Each toxin was purified from crude venom by a combination of open-column molecular sieve chromatography and the FPLC system with ion exchange or hydrophobic columns (Pharmacia, Uppsala, Sweden) as previously detailed (MIDDLEBROOK and KAISER, 1989). A assays Phospholipase assays were carried out using phosphatidylcholine as the substrate with a 2 : 1 molar ratio of Triton X-100 : phospholipid in the reaction mixture. Released fatty acids were titrated at pH 8.0 with NaOH at room temperature using a Radiometer apparatus (AIRD and KAISER, 1985). Phospholipase
ELISA
Titers were obtained by initially coating the test plates with 0.1 ml of 1 pg toxin/ml in 40 mM Na bicarbonate (pH 9.6) for a minimum of 2 hr at room temperature . After two rinses (0 .1 % Tween 20 in phosphate buffered saline, pH 7.5), dilutions of monoclonal antibody were added and incubated for 4hr (room temperature) . Plates were rinsed twice and goat anti-mouse IgG conjugated to peroxidase (Sigma Chemical Co ., St Louis, MO, U.S.A .) was added and incubated for 2 hr at 37°C . Hydrogen peroxide and chromogen (2, 2'-azino-di-[3-ethylbenzthiazoline sulfonate], Kirkegaard and Perry Laboratories, Gaithersburg, MD, U.S.A .) were added and the absorbance read at 405 nm after 1 hr (room temperature) . For biotinylated monoclonal antibody ELISA, coating of antigen was as above, followed by incubation of labeled monoclonal antibody at 0.7 pg/ml with or without the indicated concentration of unlabeled antibody . Washes, avidin-peroxidase (Sigma Chemical Co.), incubation and color generation were as above. Monoclonal antibody production Mice (BALB-C) were immunized with a mixture of pseudexins A, B and C, known as peak V (ScfanDT and MIDDLEBROOK, 1989) by the same protocol used to generate crotoxin monoclonal antibodies (KAISER and MIDDLEBROOK, 1988a). Mice with serum showing a positive ELISA to peak V were given to Dr BRIAN BUTMAN of Bionetics Inc. (Gaithersburg, MD, U.S.A .) for production of hybridomas by standard techniques under a research services contract . One fusion produced 15 positive wells, which were doubly cloned and ascites fluids produced from each line. Isotype analysis by Dr BUTMAN indicated that all were IgG, except for No . 11, which was IgM. The monoclonal antibodies were purified by Protein A-agarose (Biorad, Richmond, CA, U.S.A .) chromatography using buffers provided by the company. Biotinylation of the monoclonal antibodies Each of the affinity-purified monoclonal antibodies was biotinylated by standard techniques . Briefly, affinitypurified antibodies (1 mg/ml) were dialyzed against 0.1 M NaHC0 pH 8 .4, followed by the addition of 120,ug of biotin succinimide ester (Sigma Chemical Co .) per mg protein. After incubation at room temperature for 4 hr, the mixture was dialyzed against 50 mM Hepes (pH 7.5), 0.15 M NaCl . Concentrations of either monoclonal antibodies or rabbit IgG were determined spectrophotometrically using an extinction coefficient at 280 nm of 1 .35 for a 1 mg/ml concentration. Neutralization assays Tests for neutralizing capacity of the monoclonal antibodies were carried out as previously described (KAISER and MIDDLEBROOK, 1988a) with the mouse bioassay . Briefly, toxin and antibody were mixed and incubated for 30-60 min at room temperature before i.p. injection into groups of 15-26 g mice (female ICR). The mice were observed four days for symptoms.
Pseudexin Monoclonal Antibodies
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1 . ELISA
DosE-REsPONsE
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MONOCLONAL ANTIBODY ß-BUNGAROTOXIN .
BINDING
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Plates were coated with the specified antigen and ELISA run with all 15 monoclonal antibodies as indicated in Materials and Methods. The monoclonal antibodies had been Protein A affinitypurified and the stock concentration (negative log = 0) was 0 .75 mg/ml . RESULTS
ELISA
This work was begun before it was realized that "pseudexin" was really a mixture of isoenzymes. Thus, the immunogen used was peak V, a mixture of three pseudexin isozymes A, B and C (SCHMIDT and MIDDLEBROOK, 1989) . One fusion was performed and 15 cloned lines with positive ELISA titers to peak V were isolated . The clones were grown as ascites tumors and the resulting monoclonal antibodies purified using (NH4)2SO4 precipitation and Protein A-affinity chromatography . Concurrently, pseudexins A, B and C were successfully separated (SC1-nKIDT and MIDDLEBROOK, 1989) and were used individually for the rest of the experiments reported here. After affinity purification, the ELISA reactions of each monoclonal antibody with all three pseudexins and 14 other PLA2 toxins and enzymes were analyzed. Figure l shows examples of the typical titration curves
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TABLE I. ELISA TITERS OF PSEUDEXIN MONOCLONAL ANTIBODY REACTIONS WITH PHOSPHOLIPASE A Z TOXINS
Toxin
Pseudexin A Pseudexin B Pseudexin C Ammodytoxin ß-Bungarotoxin Taipoxin Textilotoxin N. n . atra PLA 2 Notexin Porcine PLA2 Mojave toxin Crotoxin Concolor toxin Caudoxin
Monoclonal antibody No . 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
4 .3 4 .0 4 .4 0 .0 1 .3 0 .0 0 .0 0 .0 1 .7 0 .0 0 .0 0 .0 0 .0 0 .0
4 .7 5 .3 4 .8 1 .9 4 .7 4 .5 3 .3 4 .0 4 .9 4 .6 2 .2 3 .0 2 .6 2 .0
3 .1 3 .7 5 .0 0 .1 2 .4 0 .0 0 .0 0 .0 3 .6 1 .6 0 .0 0 .0 0 .0 0 .0
3 .2 5 .1 3 .8 0 .0 4 .9 1 .7 1 .6 1 .2 5 .0 1 .9 1 .3 1 .1 0 .0 0 .0
4 .3 5 .2 5 .4 0 .0 4 .9 4 .4 4 .2 1 .4 4 .2 3 .6 1 .6 1 .7 1 .7 1 .3
3 .2 2 .4 2 .3 0 .0 3 .1 0 .0 1 .7 1 .4 2 .7 1 .7 1 .4 1 .2 0 .0 0 .0
4 .8 4 .9 5 .0 0 .0 0 .0 1 .2 1 .6 0 .0 4 .6 0 .0 0 .0 0 .0 0 .0 0 .0
4 .9 5 .2 4 .4 0 .0 0 .0 0 .0 0 .0 0 .0 4 .8 0 .0 0 .0 0 .0 0 .0 0 .0
0 .0 4 .3 4 .2 0 .0 4 .5 3 .8 1 .2 2 .4 4 .8 2 .7 0 .0 0 .0 0 .0 3 .2
3 .4 4 .8 3 .8 0 .0 4 .9 1 .4 1 .3 1 .3 5 .0 1 .1 1 .2 1 .1 0 .0 1 .2
3 .2 5 .2 3 .7 0 .0 4 .4 1 .5 1 .5 1 .3 5 .5 1 .4 1 .2 1 .1 1 .1 1 .3
2 .3 5 .3 4 .2 1 .7 1 .7 1 .8 1 .7 2 .0 2 .1 1 .8 1 .8 1 .9 2 .0 1 .6
4.3 5.3 5.1 1 .2 4.4 4.2 4.4 1 .3 4 .5 4 .1 1 .2 1 .1 1 .2 0 .0
5.0 5.4 5.3 2.0 4.4 4.5 4.4 2 .1 4 .9 4 .2 2 .4 2 .7 2 .8 2 .2
5 .4 4.2 4.5 0.0 1 .7 1 .4 1 .5 0.0 1 .7 1 .0 0.0 0.0 0.0 1 .1
Values are the negative logarithms of the dilutions giving l absorbance unit in the ELISA described in Materials and Methods . Stock concentration of each monoclonal antibody was 0 .75 mg/ml .
obtained with two toxins and all 15 of the monoclonal antibodies. With one of the immunizing antigens, pseudexin C, each of the monoclonal antibodies reacted and all but one of the curves fell at the high end or right side of the graph. In the case of a related toxin, ß-bungarotoxin, the same 15 monoclonal antibodies presented more of a spectrum of reactivities . Some of the antibodies reacted strongly, but there were a similar number that were weak reactors . In order to present the data from 210 titration curves in a more interpretable form, each curve was analyzed graphically to determine the dilution of antibody that produced an absorbance of 1 (Table 1). Thus, for Fig. 1, the estimated negative log of the dilution for the weakest reacting antibody with pseudexin C would be 2.1 or slightly more than a 100-fold dilution . Control assays, using monoclonal antibodies against unrelated antigens, presented absorbances no greater than 1 .0 at dilutions of 10', a value regarded as background for this ELISA. Therefore, any titration with pseudexin monoclonal antibodies failing to produce an absorbance of 1 or greater at 10' dilution was tabulated at 0, even though there was observable color. All of the values thus obtained are presented in Table 1 . Generally, the monoclonal antibodies recognized all three pseudexin isozymes, with number 9 representing a notable exception (Table 1). In some cases, antibody binding was essentially equivalent with each pseudexin isozyme, for example, numbers 1 and 7. On the other hand, several monoclonal antibodies differed by two to three orders of magnitude in their binding to the different pseudexin isozymes, i.e. numbers 3, 4 and 12. There was a spectrum of specificities in the recognition patterns shown by the monoclonal antibodies for the other phospholipases tested . Monoclonal antibody 8, for example, recognized all three pseudexins and notexin, but none of the other phospholipases . Others, such as 2, 5, 12, 13 and 14 bound to some extent to all the toxins or enzymes tested . Still others exhibited an intermediate pattern wherein some of the other PLA Z s were recognized (3 and 7). As judged by the number of monoclonal antibodies recognizing a given PLAZ and the relative intensities of the reactions, the nearest immunological neighbor to the pseudexins is notexin . In some instances, the antibody apparently bound better to notexin than to
Pseudexin Monoclonal Antibodies
36 3
m
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Molar ratio of antlbody :toxln
m
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Molar ratio of antibody:toxln FIG. 2 . MONOCLONAL ANTIBODY NEUTRALIZATION OF THE LETHALITY OF FSEUDEXIN3 IN MICE.
Toxins and the indicated monoclonal antibody were preincubated and injected i.p . into mice as described in Materials and Methods . The toxin amount varied somewhat depending on the weight of the mice but was in the range of 39-50 pg per mouse for pseudexin A and 30-32pg per mouse for pseudexin B (approximately 1 .5-2 LD5o). (p), ANTIBODY 3; (Q), antibody 7; (O), antibody 8.
some of the isoforms of pseudexin, i.e. monoclonal antibodies 4, 9, 10 and 11 . The next closest immunological neighbor was ß-bungarotoxin, followed by the trio of taipoxin, textilotoxin and porcine pancreatic phospholipase AZ. The PLA2 least related to the pseudexins appears to be ammodytoxin. It is also apparent that none of the crotalid or viperid toxins was recognized well by the pseudexin monoclonal antibodies. Antibody effects on toxicity and enzymatic activity
To determine whether any of the monoclonal antibodies would neutralize the lethal effects of pseudexin, antibody and toxin were mixed and injected into mice . All antibodies were tested, but only three (numbers 3, 7 and 8) demonstrated a potential for neutralization. The relative efficacy of each neutralizing antibody was determined by testing serial dilutions for their ability to protect mice from a dose of toxin producing 100% lethality (approximately 2 LD.). As shown by the data in Fig. 2, the three protective antibodies had
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v0 v v
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FIG.
3.
2
EFFECT OF MONOCLONAL ANTIBODIES ON THE PHOBPHOLIPASE A PSEUDEXIN A .
ENZYMATIC ACTIVITY OF
Toxin alone (5 jug) or toxin plus monoclonal antibody (1001ig) was preincubated at room temperature for 30 min and then added to the reaction mixture (final volume l ml). The production of acid was followed for 16 min as described in Materials and Methods . Controls show the mean and standard errors for five replicate runs carried out at various times during the course of the experiment .
essentially the same neutralization potential against both pseudexins A and B. At antibody : toxin molar ratios of approximately 0.5 and below, no neutralization was obtained. At a ratio of about 1 or above, protection was observed. Within the limits of the mouse bioassay and the dose increments used, it appears that no substantial differences exist in the neutralization efficacies of these three monoclonal antibodies for pseudexins A and B. All three of the neutralizing monoclonal antibodies were also tested for their ability to neutralize other elapid PLAZ toxins . None protected mice from 2 LD 50 of taipoxin, textilotoxin, ß-bungarotoxin or notexin. However, number 7 consistently prolonged the time to death with notexin. In several experiments, all control animals died from the challenge with 2 LD_, of notexin within 12 hr, while animals receiving toxin and antibody survived a minimum of 36 hr. The effects of all monoclonal antibodies on the enzymatic activities of the pseudexins were evaluated. It was found that antibodies 3, 7 and 8 strongly inhibited the in vitro enzymatic activities of both pseudexin A (Fig. 3) and B (data not shown) . Antibodies 1 and 15 induced a partial inhibition of the enzymatic activity of both pseudexins (Fig. 3 and data not shown), while the remainder of antibodies were without effect . The same experiments were performed with notexin with the result that 3, 7 and 8 strongly inhibited its enzymatic activity, but none of the other antibodies (including 1 and 15) brought about measurable changes (data not shown) . Competition of antibodies for binding to pseudexin
Each monoclonal antibody was labeled with biotin, and ELISA-based competition experiments performed using both pseudexins A and B and all 15 monoclonal antibodies. Typical results of such experiments are shown by the data in Fig. 4. Basically, four different patterns of behavior were observed : (a) No competition; this is seen (top frame) where up to a 50-fold molar excess of antibody 3 had no effect on the binding of labeled
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Pseudexin Monoclonal Antibodies
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10
20
30
40
50
60
Ratio of competitor antibody :labeled antibody
a
Antibody 3
a e ô >r c ô c _o U . 0 -f 0
10
20
30
40
50
60 I
Ratio of competitor anti body :labeled antibody FIG . 4. DOSE RESPONSE OF MONOCLONAL ANTIBODY comPErmoN POR BIOTINYLATED MONOCLONAL ANTIBODY BINDING TO PSEUDExrN A . Plates were coated with pseudexin A as for a regular ELISA. Biotinylated antibody with or without the stipulated excess of unlabeled monoclonal antibody was added and incubations carried out as described in Materials and Methods. Top frame: p, antibody 14 ; p, antibody 2; Q, antibody 3; 0, antibody 5; /, antibody 7; AL, antibody 8; -- A-- , antibody 13. Bottom frame : Q, antibody 3; p, antibody l ; p, antibody 7; 0, antibody 8; 0, antibody 15 .
antibody 14. (b) Full competition; this pattern is demonstrated by antibodies 2, 5 and 13 with labeled antibody 14 (top frame) or by antibodies 7 and 8 with labeled antibody 3 (bottom frame) . In full competition, the competing antibodies were as effective in blocking the binding as was the homologous unlabeled antibody . (c) Partial competition; in this pattern, competition was observed, but the maximal level achieved was a fraction, usually half, that observed in full competition. Examples of partial competition are antibodies 7 and 8 with labeled antibody 14 (top frame, Fig. 4). (d) Augmentation; this pattern is seen in the bottom frame of Fig. 4 with antibodies 1 and 15 and labeled antibody 3. Here, the presence of competitor antibody produced an increase in the binding of labeled antibody to pseudexin. It was apparent from the data in Fig. 4 and several similar experiments that it was unnecessary to perform a full titration to determine which of the four patterns of
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J. L. MIDDLEBROOK TABLE 2 . CROSS-COMPETITION OF MONOCLONAL ANTIBODY BINDING TO PSEUDEXIN
A
Competing antibody` 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Labeled antibodyt 1 = 2 - - = ++ 3 ++ 4 ---------------------------------------------------------------------------------------------------------
6 7
++
_
_
++
g 9 10
--------------------------------------------------------------------------------------------------------_ = _ _ _
12
---------------------------------------------------------------------------------------------------------
13 14 15
- specifies partial competition; = specifies competition comparable to homologous competition ; + specifies moderate (0-50%) increase in binding; + + specifies strong ( > 50%) increase in binding; ---- insufficient signal from the labeled antibody . 'Competing monoclonal antibody was added at a 50-fold molar excess over the labeled monoclonal antibody. tEach monoclonal antibody was labeled with biotin as indicated in Materials and Methods and the ELISA tests run as specified in the legend to Fig. 4.
TABLE
3.
CROSS-COMPETITION OF MONOCLONAL ANTIBODY BINDING TO PSELTDEXIN B
Competing antibody' 6 7 8 9 10 11 12 13 14 15 Labeled antibodyt 1 2 3 4 5 --------------------------------------------------------------------------------------------------------1 2 _ = ++ 3 ++ 6 7 8
--------------------------------------------------------------------------------------------------------=
-
-
12
13 14 15
- specifies partial competition ; = specifies competition comparable to homologous competition; + specifies moderate (0-50%) increase in binding; + + specifies strong ( > 50%) increase in binding; ---- insufficient signal from the labeled antibody . *Competing monoclonal antibody was added at a 50-fold molar excess over the labeled monoclonal antibody . tEach monoclonal antibody was labeled with biotin as indicated in Materials and Methods and the ELISA tests run as specified in the legend to Fig. 4.
competition occurred with a given antibody . Thus, a full matrix analysis of competition by all 15 monoclonal antibodies was performed using only a 50-fold excess of competitor. The results are tabulated in Tables 2 and 3; several conclusions or patterns can be derived
Pseudexin Monoclonal Antibodies
36 7
120 100
20 0
1
2
3
4 5 8 7 8 e 10 11 Monoclonal antibody number
12
13
14
15
FIG . 5 . MONOCLONAL ANTIBODY COMPETITION FOR RABBIT IgG BINDING TO PSEUDEXINs A AND B . Plates were coated with pseudexin A (solid) or B (diagonal lines) followed by the addition of biotinylated rabbit IgG (Protein A affinity-purified) with or without a 50-fold excess of the stipulated monoclonal antibody . The ELISA was run as in Materials and Methods, and the absorbance values reported as % of control. Values are means of nine values with standard errors .
from these tables . First, the specific patterns of competition were similar, but not identical, for pseudexins A and B. In part, this was due to the fact that certain biotinylated antibodies did not bind well enough to one of the toxins to give a dependable signal-tonoise ratio and, therefore, were not used . Specifically, labeled antibodies 4, 9 and 12 did not bind to pseudexin A, and labeled antibodies 1 and 6 did not bind to pseudexin B. Since each of these antibodies bound well to the other pseudexin isozyme (Tables 2 and 3 and data not shown), it is unlikely that the labeling procedure somehow inactivated or altered antigen recognition. Furthermore, a comparison with Table 1 indicates that antibodies 4, 9 and 12 bound poorly to pseudexin A and antibody 6 to pseudexin B in an ELISA format where the antibodies were not biotinylated . Thus, it is reasonable to suppose that the affinities of these four antibodies for either pseudexin A or B were low initially. This same explanation is probably not the case with antibody 1 and pseudexin B. In this instance, the apparent binding of antibody 1 to pseudexin B was slightly higher than that of antibody 3, and approximately the same as antibodies 9 and 15 (Table 1). A second general pattern observed was that, in a majority of cases, there was reciprocity in competition. For example, if unlabeled antibody 10 competed for the binding of labeled antibody 4 to pseudexin B, then unlabeled antibody 4 competed for the binding of labeled antibody 10 (Table 3). On the other hand, antibodies 1 and 15 produced puzzling data in that they did not compete for themselves, perhaps indicating a lack of specificity in antigen binding. On the other hand, antibodies 1 and 15 induced an increase in the binding of certain other antibodies, namely 3 and 7 (Tables 2 and 3). Since it is extremely unlikely that the binding of antibodies led to an increase in sites for antibodies 3 and 7, it seems reasonable to conclude binding of the former leads to an increase in affinity of the latter antibodies for pseudexin. To determine what effects monoclonal antibodies might have on the binding of polyclonal antibodies to all epitopes on pseudexin, IgG was affinity-purified from rabbit antisera raised to peak V (MIDDLEBROOK and KAISER, 1989) using Protein A. The IgG was then biotinylated and a competition ELISA run as above. Results (Fig. 5) indicated that some, but not all, monoclonal antibodies blocked the binding of polyclonal IgG to
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J . L . MIDDLEBROOK
pseudexin. The maximum inhibition of polyclonal IgG binding to pseudexin was induced by monoclonal antibodies 7 and 8 and amounted to 45-50% . Nine more monoclonal antibodies inhibited the binding between 20 and 40%, namely numbers 1,2,3,6 and 15 with pseudexin A and numbers 2,3,5,9,13 and 14 with pseudexin B. There was a rough correlation of apparent binding affinity and inhibition of polyclonal IgG binding, as can be seen by comparison with Table 1. Of those 15 monoclonal antibodies whose ELISA titers were 4 .5 or greater (Table 1), 11 inhibited binding 10% or more. On the other hand, the remaining four monoclonal antibodies had little or no effect on polyclonal IgG binding. When all 15 monoclonal antibodies were mixed in equal parts and used in a competition assay against the labeled rabbit IgG, maximal inhibition with pseudexin A was 60% and with pseudexin B, 55% . Thus, the mixture of all 15 monoclonal antibodies was only slightly better at competition than were antibodies 7 or 8 alone. DISCUSSION
This work was originally undertaken to develop a monoclonal antibody suitable for the detection of elapid PLAZ neurotoxins. Several of the antibodies that arose were acceptable for that purpose, including numbers 2, 5, 9, 13 and 14. In addition, the number and diversity of antibodies obtained provided a series of probes that proved useful for an immunochemical study of pseudexin and other elapid phospholipase AZ neurotoxins. Clearly, this library of monoclonal antibodies is directed to a multiplicity of epitopes on pseudexin and the relationships among antibodies are complex. A careful inspection of the data in Tables 1-3 leads one to the conclusion that some of the monoclonal antibodies are similar, if not identical . Specifically, numbers 1 and 15, 5, 13 and 14 and 10 and I I exhibit properties which are so close, that they may have come from daughter cells descended from the same clone. Even so, that leaves 11 distinct monoclonal antibodies directed to a like number of epitopes on pseudexin. It is interesting to speculate on the total number of epitopes that would be expected on a protein molecule the size of pseudexin. One possible way of obtaining such an estimate is to combine all of the monoclonal antibodies, and determine what fraction of polyclonal IgG binding was inhibited by the mixture. Then, by an obvious extrapolation, the total number of epitopes could be calculated . However, the monoclonal antibody mixture competed only slightly better than did one or the other of the individual antibodies. In other words, individual antibodies such as numbers 7 or 8 compete for 50% of the total IgG binding (Fig. 5), yet there must be more than two epitopes on the molecule . There are at least two explanations for this behavior. First, antibodies of mol . wt 150,000 are obviously much larger than a protein of pseudexin's size (13,000). Thus, binding to only one epitope on the toxin could lead to serious steric problems for the binding of additional antibodies to other epitopes . This was the result obtained by MEIJER et al. (1978) wherein it was observed that a maximum of three Fab fragments could bind to pancreatic PLAZ. A second possibility is that binding of a certain antibody to its epitope may induce conformational changes in the antigen and render many other epitopes unrecognizable to additional antibodies, thus preventing their binding. This second possibility could well explain the observation that antibodies with apparently similar affinities for pseudexin (Table 1), 7 vs 14 with pseudexin A, had quite different effects on IgG binding (Fig. 5). The fact that five monoclonal antibodies recognized all the elapid PLAZ neurotoxins tested indicates that these toxins share one or more epitopes . This conclusion confirms earlier work with rabbit antisera, wherein extensive ELISA cross-reactions were observed
Pseudexin Monoclonal Antibodies
369
within the elapid group of PLAZ toxins (MIDDLEBROOK and KAISER, 1989) . It is also in agreement with a recent study where a monoclonal antibody against notexin cross-reacted with several elapid phospholipases or other components in the venoms of elapids (MoLLIER et al., 1990). However, the spectrum of cross-reactivities exhibited by that notexin-derived monoclonal antibody suggests that it is different than any antibodies reported here . Another conclusion from the rabbit antisera study (MIDDLEBROOK and KAISER, 1989) was that notexin appears to be the nearest immunological neighbor to pseudexin. Again with the monoclonal antibodies (Table 1), the same conclusion was reached here . Furthermore, by aligning the sequence for notexin (HALPERT and ERKER, 1975) with those for pseudexins (SCHMIDT and MIDDLEBROOK, 1989), it is possible to speculate as to what region of the molecules were recognized (at least in part) by individual antibodies. Specifically, antibodies 4, 9, 10 and 11 all bind strongly to notexin and pseudexin B, but much weaker to pseudexin A. If one identifies regions where the sequences of pseudexin B and notexin are identical, but pseudexin A differs, these locations are candidates for the binding sites of monoclonal antibodies 4, 9, 10 and 11 . There are eight such regions located at residues 6, 18, 24, 46 and 47, 50, 54, 73 and 110 of pseudexin B. Three of the monoclonal antibodies (numbers 3, 7 and 8) neutralized both pseudexin A and B in a mouse lethality assay (Fig. 2). Full neutralization was observed at an antibodyto-toxin ratio of approximately 1 . This suggests that the antibodies are efficient at neutralization . Moreover, this ratio is close to the value of 1 .6 determined for the neutralization of crotoxin by a monoclonal antibody raised against its basic chain (KAISER and MIDDLEBROOK, 1989) . However, monoclonal antibodies that neutralize the lethality of phospholipases A are not easy to obtain . Of several other studies where monoclonal antibodies were produced against this class of presynaptic neurotoxin, none reported the isolation of a truly neutralizing antibody . STRONG et al. (1984) and DANSE and KEMPF (1989) obtained antibodies against ß-bungarotoxin which delayed the time to death. On the other hand, MOLLIER et al. (1990) and RAEL et al. (1986), who produced monoclonal antibodies against notexin and Mojave toxin, respectively, did not report any that would neutralize lethality. In the two studies from this laboratory, one out of four antibodies (KAISER and MIDDLEBROOK, 1989) and three out of 15 (this work) were fully neutralizing . Thus, neutralizing monoclonal antibodies are rarities, but why is not clear. The same three antibodies that neutralized lethality (3, 7 and 8) were strong inhibitors of the enzymatic activity of pseudexin (Fig. 3). Two other antibodies (1 and 15) were partial inhibitors of pseudexin's enzymatic activity, but were without measurable effect on lethality. While these data might suggest a direct link between lethality and enzymatic activity, further observations are inconsistent with such a conclusion . Antibodies 3, 7 and 8 were equally effective at inhibiting the enzymatic activities of notexin and pseudexin, yet none of the same antibodies neutralized the lethality of notexin. Moreover, in work with crotoxin monoclonal antibodies, one antibody was obtained that potently inhibited the enzymatic activity of crotoxin, but had no effect on lethality (KAISER and MIDDLEBROOK, 19886) . One unifying hypothesis which explains all these phenomena is the possibility that binding of neutralizing monoclonal antibodies leads to a major conformational change in the toxin which alters both the toxic and enzymatic sites. There does appear to be an interaction between the complete (antibodies 3, 7 and 8) and partial (antibodies 1 and 15) inhibitors of pseudexin's enzymatic activity, but the relationship is complex. For example, excess, unlabeled 3, 7 and 8 competed for themselves in binding to pseudexin (Tables 2 and 3). However, the same was not true for antibody 15,
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which suggests a non-specific type of interaction. Nevertheless, the binding of antibody 15 to pseudexin was as tight or, in the case of antibody 3, tighter than the complete inhibitors (Table 1). Acknowledgement-I am grateful to HERB GREEN and ALAN WRIGHT for their excellent technical assistance in this work . REFERENCES AIRD, S. D. and KAISER, I. I. (1985) Comparative studies on three rattlesnake toxins . Toxicon 23, 361-374. CHANG, C. C. (1985) Neurotoxins with phospholipase A, activity in snake venoms . Proc. Natn . Sci. Counc. B. ROC 9, 126-142. DANSE, J.M. and KEMPF, J. (1989) Preparation and characterization of monoclonal antibodies against ßbungarotoxin and its A- and B-chains . Toxicon 27, 1011-1019 . HALPERT, J. and EAKER, D. (1975) Amino acid sequence of a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake) . J. biol. Chem. 250, 6990-6997 . KAISER, I. 1. and MIDDLEBROOK, J. L. (1988a) Preparation of a crotoxin neutralizing monoclonal antibody . Toxicon 26, 855-865. KAISER, I. I. and MIDDLEBROOK, J. L. (1988b) Comparative properties of a neutralizing and three nonneutralizing monoclonal antibodies to crotoxin . In: Neurotoxins in Neurochemistry, pp . 43-51 (DOLLY, O. J., Ed .) . New York: John Wiley and Sons . MELIER, H., MEDDENs, J. M., DuKMAN, R., SLOTeoom, A. J. and DE HAAS, G. H. (1978) Immunological studies on pancreatic phospholipase A, : Antigenic characterization of the NH,terminal region. J. biol. Chem . 253, 8564-8569. MIDDLEBROOK, J. L. and KAISER, 1. I. (1989) Immunological relationships of phospholipase A, neurotoxins from snake venoms . Toxicon 27, 965-977. MOLLff.R, P., CHwerzoFF, S. and MENEz, A. (1990) A monoclonal antibody recognizing a conserved epitope in a group of phospholipases A, . Malec. lmmunol. 27, 7-15 . RAEL, E. D., SALO, R. J. and ZEPEDA, H. (1986) Monoclonal antibodies to Mojave toxin and use for isolation of cross-reacting proteins in Crotalus venoms . Toxicon 24, 661-668 . ROSENBERG, P. (1986) The relationship between enzymatic activity and pharmacological properties of phospholipases in natural poisons. In : Natural Toxins, pp . 129-174 (HARRIS, J. B., Ed .). Oxford : Clarendon Press. SCHMIDT, J. J. and MWDLEBROOK, J. L. (1989) Purification, sequencing and characterization of pseudexin phospholipases A, from Pseudechis porphyriacus (Australian red-bellied black snake). Toxicon 27, 805-818. STRONG, P . N., WOOD, J . N. and IvANYI, J. (1984) Characterization of monoclonal antibodies against ßbungarotoxin and their use as structural probes for related phospholipase A, enzymes and presynaptic phospholipase neurotoxins. Eur. J. Biochem. 142, 145-151 . TYLER, M. I., BARNETT, D., NICHOLSON, P., SPENCE, I. and HowDEN, M. E. H. (1987) Studies on the subunit structure of textilotoxin, a potent neurotoxin from the venom of the Australian common brown snake (Pseudonaja textilis) . Biochim. biophys. Acta 915, 210-216.
Toxkm Vol. 29, No. 3, pp. 359-370, 1991 . Printed in GICat Britain .
PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST PSEUDEXIN JOHN L . MIDDLEBROOK Department of Toxinology, Pathology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21701-5011, U.S .A . (Received 12 lute 1990; accepted for publication 27 September 1990)
J. L. MIDDLEBROOK . Preparation and characterization of monoclonal antibodies against pseudexin. Toxicon 29, 359-370, 1991 .-Fifteen hybridoma cell lines secreting monoclonal antibodies against pseudexin were developed. The cell lines were grown as ascites tumors and the resulting antibodies were purified by Protein A amity-chromatography. Several of the antibodies exhibited extensive ELISA cross-reactions with different phospholipase A2 toxins from various snake venoms, while other of the antibodies reacted only with the pseudexins . Three of the antibodies neutralized pseudexin A and B, but none of the 10 other phospholipase A2 toxins tested . These same three antibodies inhibited the enzymatic activity of pseudexin A and B and also that of notexin. After each antibody was labeled with biotin, competition experiments were carried out to determine the binding relationships among the antibodies and the pseudexins . Competitions were frequently observed, with a low of zero to a high of eight out of the 14 possibilities. Competition experiments were also carried out with biotin-labeled rabbit IgG against the pseudexins . Some of the monoclonal antibodies had no effect on rabbit IgG binding to pseudexin, while others blocked up to 50% of the binding. INTRODUCTION
A2 (PLA2) neurotoxins are a group of structurally diverse proteins found in the venoms of crotalids, viperids and elapids. In most cases, the PLA2 toxins are the most toxic components in the venoms and probably play a major role in lethality due to snake bites. As determined in mice, PLA2 LD,0 s range from 1 yg/kg for textilotoxin (TYLER et al., 1987) to 1250 pg/kg for pseudexin A (SCHMIDT and MIDDLEBROOK, 1989). Structurally, the most complex PLA2 neurotoxin is textilotoxin with five subunits, while the simplest toxins are single chain entities, such as notexin, caudoxin or pseudexin (CHANG, 1985). Each member of this class of toxins expresses a phospholipase A2 activity, although the role enzymatic activity plays in neurotoxicity is controversial (ROSENBERG, 1986). To better understand the structural relationships among the PLA2 toxins, we have been studying their immunochemistries. We determined that extensive ELISA cross-reactions can be observed with polyclonal antisera raised to the purified neurotoxins (MIDDLEBROOK and KAISER, 1989). Based on intensities of the cross reactions, it was possible to assign each PLA2 neurotoxin to one of two serological classes: (a) elapid and viperid or (b) crotalid. The same data are also useful for determining immunological similarity of a PHOSPHOLIPASE
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given two toxins or suggest the possibility of common, neutralizing epitopes on the toxins (MIDDLEBROOK, in preparation). Previous studies showed that it was possible to obtain a monoclonal antibody that would recognize all of the crotalid neurotoxins tested KAISER and MIDDLEBROOK, 1988a) . In order to develop a similar reagent for the elapid/viperid class of PLAZ neurotoxins, monoclonal antibodies have been raised to several toxins in this group. One fusion, using pseudexin as the antigen, produced 15 clones. Properties of the monoclonal antibodies elaborated by these clones are reported here . MATERIALS AND METHODS Toxins Each toxin was purified from crude venom by a combination of open-column molecular sieve chromatography and the FPLC system with ion exchange or hydrophobic columns (Pharmacia, Uppsala, Sweden) as previously detailed (MIDDLEBROOK and KAISER, 1989). A assays Phospholipase assays were carried out using phosphatidylcholine as the substrate with a 2 : 1 molar ratio of Triton X-100 : phospholipid in the reaction mixture. Released fatty acids were titrated at pH 8.0 with NaOH at room temperature using a Radiometer apparatus (AIRD and KAISER, 1985). Phospholipase
ELISA
Titers were obtained by initially coating the test plates with 0.1 ml of 1 pg toxin/ml in 40 mM Na bicarbonate (pH 9.6) for a minimum of 2 hr at room temperature . After two rinses (0 .1 % Tween 20 in phosphate buffered saline, pH 7.5), dilutions of monoclonal antibody were added and incubated for 4hr (room temperature) . Plates were rinsed twice and goat anti-mouse IgG conjugated to peroxidase (Sigma Chemical Co ., St Louis, MO, U.S.A .) was added and incubated for 2 hr at 37°C . Hydrogen peroxide and chromogen (2, 2'-azino-di-[3-ethylbenzthiazoline sulfonate], Kirkegaard and Perry Laboratories, Gaithersburg, MD, U.S.A .) were added and the absorbance read at 405 nm after 1 hr (room temperature) . For biotinylated monoclonal antibody ELISA, coating of antigen was as above, followed by incubation of labeled monoclonal antibody at 0.7 pg/ml with or without the indicated concentration of unlabeled antibody . Washes, avidin-peroxidase (Sigma Chemical Co.), incubation and color generation were as above. Monoclonal antibody production Mice (BALB-C) were immunized with a mixture of pseudexins A, B and C, known as peak V (ScfanDT and MIDDLEBROOK, 1989) by the same protocol used to generate crotoxin monoclonal antibodies (KAISER and MIDDLEBROOK, 1988a). Mice with serum showing a positive ELISA to peak V were given to Dr BRIAN BUTMAN of Bionetics Inc. (Gaithersburg, MD, U.S.A .) for production of hybridomas by standard techniques under a research services contract . One fusion produced 15 positive wells, which were doubly cloned and ascites fluids produced from each line. Isotype analysis by Dr BUTMAN indicated that all were IgG, except for No . 11, which was IgM. The monoclonal antibodies were purified by Protein A-agarose (Biorad, Richmond, CA, U.S.A .) chromatography using buffers provided by the company. Biotinylation of the monoclonal antibodies Each of the affinity-purified monoclonal antibodies was biotinylated by standard techniques . Briefly, affinitypurified antibodies (1 mg/ml) were dialyzed against 0.1 M NaHC0 pH 8 .4, followed by the addition of 120,ug of biotin succinimide ester (Sigma Chemical Co .) per mg protein. After incubation at room temperature for 4 hr, the mixture was dialyzed against 50 mM Hepes (pH 7.5), 0.15 M NaCl . Concentrations of either monoclonal antibodies or rabbit IgG were determined spectrophotometrically using an extinction coefficient at 280 nm of 1 .35 for a 1 mg/ml concentration. Neutralization assays Tests for neutralizing capacity of the monoclonal antibodies were carried out as previously described (KAISER and MIDDLEBROOK, 1988a) with the mouse bioassay . Briefly, toxin and antibody were mixed and incubated for 30-60 min at room temperature before i.p. injection into groups of 15-26 g mice (female ICR). The mice were observed four days for symptoms.
Pseudexin Monoclonal Antibodies
361
Pseudexin C
E e
N 0 v
s 8e e a
8-bungarotoxin
E
e é 0 A a
a
FIG .
1 . ELISA
DosE-REsPONsE
OF
MONOCLONAL ANTIBODY ß-BUNGAROTOXIN .
BINDING
TO
PSEUDE)QN
C
AND
Plates were coated with the specified antigen and ELISA run with all 15 monoclonal antibodies as indicated in Materials and Methods. The monoclonal antibodies had been Protein A affinitypurified and the stock concentration (negative log = 0) was 0 .75 mg/ml . RESULTS
ELISA
This work was begun before it was realized that "pseudexin" was really a mixture of isoenzymes. Thus, the immunogen used was peak V, a mixture of three pseudexin isozymes A, B and C (SCHMIDT and MIDDLEBROOK, 1989) . One fusion was performed and 15 cloned lines with positive ELISA titers to peak V were isolated . The clones were grown as ascites tumors and the resulting monoclonal antibodies purified using (NH4)2SO4 precipitation and Protein A-affinity chromatography . Concurrently, pseudexins A, B and C were successfully separated (SC1-nKIDT and MIDDLEBROOK, 1989) and were used individually for the rest of the experiments reported here. After affinity purification, the ELISA reactions of each monoclonal antibody with all three pseudexins and 14 other PLA2 toxins and enzymes were analyzed. Figure l shows examples of the typical titration curves
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TABLE I. ELISA TITERS OF PSEUDEXIN MONOCLONAL ANTIBODY REACTIONS WITH PHOSPHOLIPASE A Z TOXINS
Toxin
Pseudexin A Pseudexin B Pseudexin C Ammodytoxin ß-Bungarotoxin Taipoxin Textilotoxin N. n . atra PLA 2 Notexin Porcine PLA2 Mojave toxin Crotoxin Concolor toxin Caudoxin
Monoclonal antibody No . 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
4 .3 4 .0 4 .4 0 .0 1 .3 0 .0 0 .0 0 .0 1 .7 0 .0 0 .0 0 .0 0 .0 0 .0
4 .7 5 .3 4 .8 1 .9 4 .7 4 .5 3 .3 4 .0 4 .9 4 .6 2 .2 3 .0 2 .6 2 .0
3 .1 3 .7 5 .0 0 .1 2 .4 0 .0 0 .0 0 .0 3 .6 1 .6 0 .0 0 .0 0 .0 0 .0
3 .2 5 .1 3 .8 0 .0 4 .9 1 .7 1 .6 1 .2 5 .0 1 .9 1 .3 1 .1 0 .0 0 .0
4 .3 5 .2 5 .4 0 .0 4 .9 4 .4 4 .2 1 .4 4 .2 3 .6 1 .6 1 .7 1 .7 1 .3
3 .2 2 .4 2 .3 0 .0 3 .1 0 .0 1 .7 1 .4 2 .7 1 .7 1 .4 1 .2 0 .0 0 .0
4 .8 4 .9 5 .0 0 .0 0 .0 1 .2 1 .6 0 .0 4 .6 0 .0 0 .0 0 .0 0 .0 0 .0
4 .9 5 .2 4 .4 0 .0 0 .0 0 .0 0 .0 0 .0 4 .8 0 .0 0 .0 0 .0 0 .0 0 .0
0 .0 4 .3 4 .2 0 .0 4 .5 3 .8 1 .2 2 .4 4 .8 2 .7 0 .0 0 .0 0 .0 3 .2
3 .4 4 .8 3 .8 0 .0 4 .9 1 .4 1 .3 1 .3 5 .0 1 .1 1 .2 1 .1 0 .0 1 .2
3 .2 5 .2 3 .7 0 .0 4 .4 1 .5 1 .5 1 .3 5 .5 1 .4 1 .2 1 .1 1 .1 1 .3
2 .3 5 .3 4 .2 1 .7 1 .7 1 .8 1 .7 2 .0 2 .1 1 .8 1 .8 1 .9 2 .0 1 .6
4.3 5.3 5.1 1 .2 4.4 4.2 4.4 1 .3 4 .5 4 .1 1 .2 1 .1 1 .2 0 .0
5.0 5.4 5.3 2.0 4.4 4.5 4.4 2 .1 4 .9 4 .2 2 .4 2 .7 2 .8 2 .2
5 .4 4.2 4.5 0.0 1 .7 1 .4 1 .5 0.0 1 .7 1 .0 0.0 0.0 0.0 1 .1
Values are the negative logarithms of the dilutions giving l absorbance unit in the ELISA described in Materials and Methods . Stock concentration of each monoclonal antibody was 0 .75 mg/ml .
obtained with two toxins and all 15 of the monoclonal antibodies. With one of the immunizing antigens, pseudexin C, each of the monoclonal antibodies reacted and all but one of the curves fell at the high end or right side of the graph. In the case of a related toxin, ß-bungarotoxin, the same 15 monoclonal antibodies presented more of a spectrum of reactivities . Some of the antibodies reacted strongly, but there were a similar number that were weak reactors . In order to present the data from 210 titration curves in a more interpretable form, each curve was analyzed graphically to determine the dilution of antibody that produced an absorbance of 1 (Table 1). Thus, for Fig. 1, the estimated negative log of the dilution for the weakest reacting antibody with pseudexin C would be 2.1 or slightly more than a 100-fold dilution . Control assays, using monoclonal antibodies against unrelated antigens, presented absorbances no greater than 1 .0 at dilutions of 10', a value regarded as background for this ELISA. Therefore, any titration with pseudexin monoclonal antibodies failing to produce an absorbance of 1 or greater at 10' dilution was tabulated at 0, even though there was observable color. All of the values thus obtained are presented in Table 1 . Generally, the monoclonal antibodies recognized all three pseudexin isozymes, with number 9 representing a notable exception (Table 1). In some cases, antibody binding was essentially equivalent with each pseudexin isozyme, for example, numbers 1 and 7. On the other hand, several monoclonal antibodies differed by two to three orders of magnitude in their binding to the different pseudexin isozymes, i.e. numbers 3, 4 and 12. There was a spectrum of specificities in the recognition patterns shown by the monoclonal antibodies for the other phospholipases tested . Monoclonal antibody 8, for example, recognized all three pseudexins and notexin, but none of the other phospholipases . Others, such as 2, 5, 12, 13 and 14 bound to some extent to all the toxins or enzymes tested . Still others exhibited an intermediate pattern wherein some of the other PLA Z s were recognized (3 and 7). As judged by the number of monoclonal antibodies recognizing a given PLAZ and the relative intensities of the reactions, the nearest immunological neighbor to the pseudexins is notexin . In some instances, the antibody apparently bound better to notexin than to
Pseudexin Monoclonal Antibodies
36 3
m
ô i:
w
Molar ratio of antlbody :toxln
m
w ô ir Co
Molar ratio of antibody:toxln FIG. 2 . MONOCLONAL ANTIBODY NEUTRALIZATION OF THE LETHALITY OF FSEUDEXIN3 IN MICE.
Toxins and the indicated monoclonal antibody were preincubated and injected i.p . into mice as described in Materials and Methods . The toxin amount varied somewhat depending on the weight of the mice but was in the range of 39-50 pg per mouse for pseudexin A and 30-32pg per mouse for pseudexin B (approximately 1 .5-2 LD5o). (p), ANTIBODY 3; (Q), antibody 7; (O), antibody 8.
some of the isoforms of pseudexin, i.e. monoclonal antibodies 4, 9, 10 and 11 . The next closest immunological neighbor was ß-bungarotoxin, followed by the trio of taipoxin, textilotoxin and porcine pancreatic phospholipase AZ. The PLA2 least related to the pseudexins appears to be ammodytoxin. It is also apparent that none of the crotalid or viperid toxins was recognized well by the pseudexin monoclonal antibodies. Antibody effects on toxicity and enzymatic activity
To determine whether any of the monoclonal antibodies would neutralize the lethal effects of pseudexin, antibody and toxin were mixed and injected into mice . All antibodies were tested, but only three (numbers 3, 7 and 8) demonstrated a potential for neutralization. The relative efficacy of each neutralizing antibody was determined by testing serial dilutions for their ability to protect mice from a dose of toxin producing 100% lethality (approximately 2 LD.). As shown by the data in Fig. 2, the three protective antibodies had
36 4
J. L . MIDDLEBROOK
v0 v v
a m 3
FIG.
3.
2
EFFECT OF MONOCLONAL ANTIBODIES ON THE PHOBPHOLIPASE A PSEUDEXIN A .
ENZYMATIC ACTIVITY OF
Toxin alone (5 jug) or toxin plus monoclonal antibody (1001ig) was preincubated at room temperature for 30 min and then added to the reaction mixture (final volume l ml). The production of acid was followed for 16 min as described in Materials and Methods . Controls show the mean and standard errors for five replicate runs carried out at various times during the course of the experiment .
essentially the same neutralization potential against both pseudexins A and B. At antibody : toxin molar ratios of approximately 0.5 and below, no neutralization was obtained. At a ratio of about 1 or above, protection was observed. Within the limits of the mouse bioassay and the dose increments used, it appears that no substantial differences exist in the neutralization efficacies of these three monoclonal antibodies for pseudexins A and B. All three of the neutralizing monoclonal antibodies were also tested for their ability to neutralize other elapid PLAZ toxins . None protected mice from 2 LD 50 of taipoxin, textilotoxin, ß-bungarotoxin or notexin. However, number 7 consistently prolonged the time to death with notexin. In several experiments, all control animals died from the challenge with 2 LD_, of notexin within 12 hr, while animals receiving toxin and antibody survived a minimum of 36 hr. The effects of all monoclonal antibodies on the enzymatic activities of the pseudexins were evaluated. It was found that antibodies 3, 7 and 8 strongly inhibited the in vitro enzymatic activities of both pseudexin A (Fig. 3) and B (data not shown) . Antibodies 1 and 15 induced a partial inhibition of the enzymatic activity of both pseudexins (Fig. 3 and data not shown), while the remainder of antibodies were without effect . The same experiments were performed with notexin with the result that 3, 7 and 8 strongly inhibited its enzymatic activity, but none of the other antibodies (including 1 and 15) brought about measurable changes (data not shown) . Competition of antibodies for binding to pseudexin
Each monoclonal antibody was labeled with biotin, and ELISA-based competition experiments performed using both pseudexins A and B and all 15 monoclonal antibodies. Typical results of such experiments are shown by the data in Fig. 4. Basically, four different patterns of behavior were observed : (a) No competition; this is seen (top frame) where up to a 50-fold molar excess of antibody 3 had no effect on the binding of labeled
365
Pseudexin Monoclonal Antibodies
8c e
s
a m ô
c
ô c _o a 0
10
20
30
40
50
60
Ratio of competitor antibody :labeled antibody
a
Antibody 3
a e ô >r c ô c _o U . 0 -f 0
10
20
30
40
50
60 I
Ratio of competitor anti body :labeled antibody FIG . 4. DOSE RESPONSE OF MONOCLONAL ANTIBODY comPErmoN POR BIOTINYLATED MONOCLONAL ANTIBODY BINDING TO PSEUDExrN A . Plates were coated with pseudexin A as for a regular ELISA. Biotinylated antibody with or without the stipulated excess of unlabeled monoclonal antibody was added and incubations carried out as described in Materials and Methods. Top frame: p, antibody 14 ; p, antibody 2; Q, antibody 3; 0, antibody 5; /, antibody 7; AL, antibody 8; -- A-- , antibody 13. Bottom frame : Q, antibody 3; p, antibody l ; p, antibody 7; 0, antibody 8; 0, antibody 15 .
antibody 14. (b) Full competition; this pattern is demonstrated by antibodies 2, 5 and 13 with labeled antibody 14 (top frame) or by antibodies 7 and 8 with labeled antibody 3 (bottom frame) . In full competition, the competing antibodies were as effective in blocking the binding as was the homologous unlabeled antibody . (c) Partial competition; in this pattern, competition was observed, but the maximal level achieved was a fraction, usually half, that observed in full competition. Examples of partial competition are antibodies 7 and 8 with labeled antibody 14 (top frame, Fig. 4). (d) Augmentation; this pattern is seen in the bottom frame of Fig. 4 with antibodies 1 and 15 and labeled antibody 3. Here, the presence of competitor antibody produced an increase in the binding of labeled antibody to pseudexin. It was apparent from the data in Fig. 4 and several similar experiments that it was unnecessary to perform a full titration to determine which of the four patterns of
36 6
J. L. MIDDLEBROOK TABLE 2 . CROSS-COMPETITION OF MONOCLONAL ANTIBODY BINDING TO PSEUDEXIN
A
Competing antibody` 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Labeled antibodyt 1 = 2 - - = ++ 3 ++ 4 ---------------------------------------------------------------------------------------------------------
6 7
++
_
_
++
g 9 10
--------------------------------------------------------------------------------------------------------_ = _ _ _
12
---------------------------------------------------------------------------------------------------------
13 14 15
- specifies partial competition; = specifies competition comparable to homologous competition ; + specifies moderate (0-50%) increase in binding; + + specifies strong ( > 50%) increase in binding; ---- insufficient signal from the labeled antibody . 'Competing monoclonal antibody was added at a 50-fold molar excess over the labeled monoclonal antibody. tEach monoclonal antibody was labeled with biotin as indicated in Materials and Methods and the ELISA tests run as specified in the legend to Fig. 4.
TABLE
3.
CROSS-COMPETITION OF MONOCLONAL ANTIBODY BINDING TO PSELTDEXIN B
Competing antibody' 6 7 8 9 10 11 12 13 14 15 Labeled antibodyt 1 2 3 4 5 --------------------------------------------------------------------------------------------------------1 2 _ = ++ 3 ++ 6 7 8
--------------------------------------------------------------------------------------------------------=
-
-
12
13 14 15
- specifies partial competition ; = specifies competition comparable to homologous competition; + specifies moderate (0-50%) increase in binding; + + specifies strong ( > 50%) increase in binding; ---- insufficient signal from the labeled antibody . *Competing monoclonal antibody was added at a 50-fold molar excess over the labeled monoclonal antibody . tEach monoclonal antibody was labeled with biotin as indicated in Materials and Methods and the ELISA tests run as specified in the legend to Fig. 4.
competition occurred with a given antibody . Thus, a full matrix analysis of competition by all 15 monoclonal antibodies was performed using only a 50-fold excess of competitor. The results are tabulated in Tables 2 and 3; several conclusions or patterns can be derived
Pseudexin Monoclonal Antibodies
36 7
120 100
20 0
1
2
3
4 5 8 7 8 e 10 11 Monoclonal antibody number
12
13
14
15
FIG . 5 . MONOCLONAL ANTIBODY COMPETITION FOR RABBIT IgG BINDING TO PSEUDEXINs A AND B . Plates were coated with pseudexin A (solid) or B (diagonal lines) followed by the addition of biotinylated rabbit IgG (Protein A affinity-purified) with or without a 50-fold excess of the stipulated monoclonal antibody . The ELISA was run as in Materials and Methods, and the absorbance values reported as % of control. Values are means of nine values with standard errors .
from these tables . First, the specific patterns of competition were similar, but not identical, for pseudexins A and B. In part, this was due to the fact that certain biotinylated antibodies did not bind well enough to one of the toxins to give a dependable signal-tonoise ratio and, therefore, were not used . Specifically, labeled antibodies 4, 9 and 12 did not bind to pseudexin A, and labeled antibodies 1 and 6 did not bind to pseudexin B. Since each of these antibodies bound well to the other pseudexin isozyme (Tables 2 and 3 and data not shown), it is unlikely that the labeling procedure somehow inactivated or altered antigen recognition. Furthermore, a comparison with Table 1 indicates that antibodies 4, 9 and 12 bound poorly to pseudexin A and antibody 6 to pseudexin B in an ELISA format where the antibodies were not biotinylated . Thus, it is reasonable to suppose that the affinities of these four antibodies for either pseudexin A or B were low initially. This same explanation is probably not the case with antibody 1 and pseudexin B. In this instance, the apparent binding of antibody 1 to pseudexin B was slightly higher than that of antibody 3, and approximately the same as antibodies 9 and 15 (Table 1). A second general pattern observed was that, in a majority of cases, there was reciprocity in competition. For example, if unlabeled antibody 10 competed for the binding of labeled antibody 4 to pseudexin B, then unlabeled antibody 4 competed for the binding of labeled antibody 10 (Table 3). On the other hand, antibodies 1 and 15 produced puzzling data in that they did not compete for themselves, perhaps indicating a lack of specificity in antigen binding. On the other hand, antibodies 1 and 15 induced an increase in the binding of certain other antibodies, namely 3 and 7 (Tables 2 and 3). Since it is extremely unlikely that the binding of antibodies led to an increase in sites for antibodies 3 and 7, it seems reasonable to conclude binding of the former leads to an increase in affinity of the latter antibodies for pseudexin. To determine what effects monoclonal antibodies might have on the binding of polyclonal antibodies to all epitopes on pseudexin, IgG was affinity-purified from rabbit antisera raised to peak V (MIDDLEBROOK and KAISER, 1989) using Protein A. The IgG was then biotinylated and a competition ELISA run as above. Results (Fig. 5) indicated that some, but not all, monoclonal antibodies blocked the binding of polyclonal IgG to
36 8
J . L . MIDDLEBROOK
pseudexin. The maximum inhibition of polyclonal IgG binding to pseudexin was induced by monoclonal antibodies 7 and 8 and amounted to 45-50% . Nine more monoclonal antibodies inhibited the binding between 20 and 40%, namely numbers 1,2,3,6 and 15 with pseudexin A and numbers 2,3,5,9,13 and 14 with pseudexin B. There was a rough correlation of apparent binding affinity and inhibition of polyclonal IgG binding, as can be seen by comparison with Table 1. Of those 15 monoclonal antibodies whose ELISA titers were 4 .5 or greater (Table 1), 11 inhibited binding 10% or more. On the other hand, the remaining four monoclonal antibodies had little or no effect on polyclonal IgG binding. When all 15 monoclonal antibodies were mixed in equal parts and used in a competition assay against the labeled rabbit IgG, maximal inhibition with pseudexin A was 60% and with pseudexin B, 55% . Thus, the mixture of all 15 monoclonal antibodies was only slightly better at competition than were antibodies 7 or 8 alone. DISCUSSION
This work was originally undertaken to develop a monoclonal antibody suitable for the detection of elapid PLAZ neurotoxins. Several of the antibodies that arose were acceptable for that purpose, including numbers 2, 5, 9, 13 and 14. In addition, the number and diversity of antibodies obtained provided a series of probes that proved useful for an immunochemical study of pseudexin and other elapid phospholipase AZ neurotoxins. Clearly, this library of monoclonal antibodies is directed to a multiplicity of epitopes on pseudexin and the relationships among antibodies are complex. A careful inspection of the data in Tables 1-3 leads one to the conclusion that some of the monoclonal antibodies are similar, if not identical . Specifically, numbers 1 and 15, 5, 13 and 14 and 10 and I I exhibit properties which are so close, that they may have come from daughter cells descended from the same clone. Even so, that leaves 11 distinct monoclonal antibodies directed to a like number of epitopes on pseudexin. It is interesting to speculate on the total number of epitopes that would be expected on a protein molecule the size of pseudexin. One possible way of obtaining such an estimate is to combine all of the monoclonal antibodies, and determine what fraction of polyclonal IgG binding was inhibited by the mixture. Then, by an obvious extrapolation, the total number of epitopes could be calculated . However, the monoclonal antibody mixture competed only slightly better than did one or the other of the individual antibodies. In other words, individual antibodies such as numbers 7 or 8 compete for 50% of the total IgG binding (Fig. 5), yet there must be more than two epitopes on the molecule . There are at least two explanations for this behavior. First, antibodies of mol . wt 150,000 are obviously much larger than a protein of pseudexin's size (13,000). Thus, binding to only one epitope on the toxin could lead to serious steric problems for the binding of additional antibodies to other epitopes . This was the result obtained by MEIJER et al. (1978) wherein it was observed that a maximum of three Fab fragments could bind to pancreatic PLAZ. A second possibility is that binding of a certain antibody to its epitope may induce conformational changes in the antigen and render many other epitopes unrecognizable to additional antibodies, thus preventing their binding. This second possibility could well explain the observation that antibodies with apparently similar affinities for pseudexin (Table 1), 7 vs 14 with pseudexin A, had quite different effects on IgG binding (Fig. 5). The fact that five monoclonal antibodies recognized all the elapid PLAZ neurotoxins tested indicates that these toxins share one or more epitopes . This conclusion confirms earlier work with rabbit antisera, wherein extensive ELISA cross-reactions were observed
Pseudexin Monoclonal Antibodies
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within the elapid group of PLAZ toxins (MIDDLEBROOK and KAISER, 1989) . It is also in agreement with a recent study where a monoclonal antibody against notexin cross-reacted with several elapid phospholipases or other components in the venoms of elapids (MoLLIER et al., 1990). However, the spectrum of cross-reactivities exhibited by that notexin-derived monoclonal antibody suggests that it is different than any antibodies reported here . Another conclusion from the rabbit antisera study (MIDDLEBROOK and KAISER, 1989) was that notexin appears to be the nearest immunological neighbor to pseudexin. Again with the monoclonal antibodies (Table 1), the same conclusion was reached here . Furthermore, by aligning the sequence for notexin (HALPERT and ERKER, 1975) with those for pseudexins (SCHMIDT and MIDDLEBROOK, 1989), it is possible to speculate as to what region of the molecules were recognized (at least in part) by individual antibodies. Specifically, antibodies 4, 9, 10 and 11 all bind strongly to notexin and pseudexin B, but much weaker to pseudexin A. If one identifies regions where the sequences of pseudexin B and notexin are identical, but pseudexin A differs, these locations are candidates for the binding sites of monoclonal antibodies 4, 9, 10 and 11 . There are eight such regions located at residues 6, 18, 24, 46 and 47, 50, 54, 73 and 110 of pseudexin B. Three of the monoclonal antibodies (numbers 3, 7 and 8) neutralized both pseudexin A and B in a mouse lethality assay (Fig. 2). Full neutralization was observed at an antibodyto-toxin ratio of approximately 1 . This suggests that the antibodies are efficient at neutralization . Moreover, this ratio is close to the value of 1 .6 determined for the neutralization of crotoxin by a monoclonal antibody raised against its basic chain (KAISER and MIDDLEBROOK, 1989) . However, monoclonal antibodies that neutralize the lethality of phospholipases A are not easy to obtain . Of several other studies where monoclonal antibodies were produced against this class of presynaptic neurotoxin, none reported the isolation of a truly neutralizing antibody . STRONG et al. (1984) and DANSE and KEMPF (1989) obtained antibodies against ß-bungarotoxin which delayed the time to death. On the other hand, MOLLIER et al. (1990) and RAEL et al. (1986), who produced monoclonal antibodies against notexin and Mojave toxin, respectively, did not report any that would neutralize lethality. In the two studies from this laboratory, one out of four antibodies (KAISER and MIDDLEBROOK, 1989) and three out of 15 (this work) were fully neutralizing . Thus, neutralizing monoclonal antibodies are rarities, but why is not clear. The same three antibodies that neutralized lethality (3, 7 and 8) were strong inhibitors of the enzymatic activity of pseudexin (Fig. 3). Two other antibodies (1 and 15) were partial inhibitors of pseudexin's enzymatic activity, but were without measurable effect on lethality. While these data might suggest a direct link between lethality and enzymatic activity, further observations are inconsistent with such a conclusion . Antibodies 3, 7 and 8 were equally effective at inhibiting the enzymatic activities of notexin and pseudexin, yet none of the same antibodies neutralized the lethality of notexin. Moreover, in work with crotoxin monoclonal antibodies, one antibody was obtained that potently inhibited the enzymatic activity of crotoxin, but had no effect on lethality (KAISER and MIDDLEBROOK, 19886) . One unifying hypothesis which explains all these phenomena is the possibility that binding of neutralizing monoclonal antibodies leads to a major conformational change in the toxin which alters both the toxic and enzymatic sites. There does appear to be an interaction between the complete (antibodies 3, 7 and 8) and partial (antibodies 1 and 15) inhibitors of pseudexin's enzymatic activity, but the relationship is complex. For example, excess, unlabeled 3, 7 and 8 competed for themselves in binding to pseudexin (Tables 2 and 3). However, the same was not true for antibody 15,
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which suggests a non-specific type of interaction. Nevertheless, the binding of antibody 15 to pseudexin was as tight or, in the case of antibody 3, tighter than the complete inhibitors (Table 1). Acknowledgement-I am grateful to HERB GREEN and ALAN WRIGHT for their excellent technical assistance in this work . REFERENCES AIRD, S. D. and KAISER, I. I. (1985) Comparative studies on three rattlesnake toxins . Toxicon 23, 361-374. CHANG, C. C. (1985) Neurotoxins with phospholipase A, activity in snake venoms . Proc. Natn . Sci. Counc. B. ROC 9, 126-142. DANSE, J.M. and KEMPF, J. (1989) Preparation and characterization of monoclonal antibodies against ßbungarotoxin and its A- and B-chains . Toxicon 27, 1011-1019 . HALPERT, J. and EAKER, D. (1975) Amino acid sequence of a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake) . J. biol. Chem. 250, 6990-6997 . KAISER, I. 1. and MIDDLEBROOK, J. L. (1988a) Preparation of a crotoxin neutralizing monoclonal antibody . Toxicon 26, 855-865. KAISER, I. I. and MIDDLEBROOK, J. L. (1988b) Comparative properties of a neutralizing and three nonneutralizing monoclonal antibodies to crotoxin . In: Neurotoxins in Neurochemistry, pp . 43-51 (DOLLY, O. J., Ed .) . New York: John Wiley and Sons . MELIER, H., MEDDENs, J. M., DuKMAN, R., SLOTeoom, A. J. and DE HAAS, G. H. (1978) Immunological studies on pancreatic phospholipase A, : Antigenic characterization of the NH,terminal region. J. biol. Chem . 253, 8564-8569. MIDDLEBROOK, J. L. and KAISER, 1. I. (1989) Immunological relationships of phospholipase A, neurotoxins from snake venoms . Toxicon 27, 965-977. MOLLff.R, P., CHwerzoFF, S. and MENEz, A. (1990) A monoclonal antibody recognizing a conserved epitope in a group of phospholipases A, . Malec. lmmunol. 27, 7-15 . RAEL, E. D., SALO, R. J. and ZEPEDA, H. (1986) Monoclonal antibodies to Mojave toxin and use for isolation of cross-reacting proteins in Crotalus venoms . Toxicon 24, 661-668 . ROSENBERG, P. (1986) The relationship between enzymatic activity and pharmacological properties of phospholipases in natural poisons. In : Natural Toxins, pp . 129-174 (HARRIS, J. B., Ed .). Oxford : Clarendon Press. SCHMIDT, J. J. and MWDLEBROOK, J. L. (1989) Purification, sequencing and characterization of pseudexin phospholipases A, from Pseudechis porphyriacus (Australian red-bellied black snake). Toxicon 27, 805-818. STRONG, P . N., WOOD, J . N. and IvANYI, J. (1984) Characterization of monoclonal antibodies against ßbungarotoxin and their use as structural probes for related phospholipase A, enzymes and presynaptic phospholipase neurotoxins. Eur. J. Biochem. 142, 145-151 . TYLER, M. I., BARNETT, D., NICHOLSON, P., SPENCE, I. and HowDEN, M. E. H. (1987) Studies on the subunit structure of textilotoxin, a potent neurotoxin from the venom of the Australian common brown snake (Pseudonaja textilis) . Biochim. biophys. Acta 915, 210-216.
