version ISSN 0104-7930
J. Venom. Anim. Toxins vol. 2 n. 2 Botucatu 1996
A SENSITIVE AND SPECIFIC IMMUNOASSAY FOR THE MEASUREMENT OF THE ANTIBODIES PRESENT IN HORSE ANTIVENOMS ENDOWED WITH THE CAPACITY TO BLOCK THE PHOSPHOLIPASE A2-DEPENDENT HEMOLYSIS INDUCED BY SNAKE VENOMS
1 Immunochemistry Laboratory, Butantan Institute, State of São Paulo, Brazil, 2 Hyperimmune Plasma Processing Service, Butantan Institute, State of São Paulo, Brazil.
ABSTRACT: Phospholipase A2 (PLA2), a component of most snake venom toxins, cleaves 3-sn-phosphoglycerides releasing lysophosphatidyl-choline. The indirect quantitative assay method for PLA2 was standardized for specific antivenom titration in a fast and sensitive assay by the similarity with the hemolysis induced by PLA2 and by complement system in sheep erythrocytes. The curves obtained by plotting the degree of hemolysis against the doses of snake venom are concave to the abscissa axis following an equation similar to that previously described for the hemolysis induced by the C system. We observed that venoms of some Bothrops, Crotalus and Micrurus species contained around 1 x 10 to 10 Z/mg of venom, while the venom of Naja contained over one million Z/mg. Antibodies against PLA2 were titrated by incubating amounts of venom predetermined to give 1 to 5 Z with various dilutions of the antivenoms, and the remaining active PLA2 was determined in the hemolytic assay. We observed the following: a) the antivenoms contained specific antibodies against the PLA2 present in the corresponding venoms; b) cross-reactivity was not detected among PLA2 epitopes from venoms and nonspecific antivenoms; and c) the assay quantitatively performed determined the specific antibodies directed to epitopes on the molecule of PLA2. The method described in this paper is highly specific, sensitive and reproducible, besides being fast and inexpensive.
KEY WORDS: PLA2, snake venoms, immunoassay, antivenoms, indirect hemolysis.
Phospholipases A2 (PLA2) cleave the 3-sn-phosphoglycerides at the (B)H-C-O-C=O-R releasing lysophosphatidyl-choline, ethanolamine, inositol or serine. PLA2 are the most commonly ones present in most snake venoms(1,7,10). They may exist either as single-chain polypeptides of 110-125 amino acids or as complexes of two or more subunits, but usually as the most toxic components of snake venoms such as taipoxin (LD50=2.0µg/Kg), notexin (LD50=17.0µg/Kg), crotoxin (LD50=25.0µg/Kg), beta-bungarotoxin (LD50=19-130 µg/Kg), and notechin II-5 (LD50=49, µg/kg) (2,18). Crotoxin, the main toxic component of Crotalus durissus terrrificus venom, is a protein composed of two tightly associated nonidentical subunits: a basic and weakly toxic component (B) carrying the phospholipase activity (PLA2), and an acidic, non-toxic component (A) (crotapotin) devoid of enzymatic activity (1,8,15,16). Component B is a single polypeptide chain of 123 amino acids presenting a great similarity with the other PLA2, while component A is composed of three polypeptides linked by several disulfide bridges (5). The PLA2 present in Bothrops jararacussu venoms has been recently isolated and characterized(12), while that present in Micrurus venoms has been recently cloned(9). Some PLA2 act presynaptically inhibiting release, synthesis, storage, or turnorver of neurotransmitters, while others damage skeletal muscles or induce indirect hemolysis through the lysolecithin formation from extra erythrocyte lecithin sources(6). Some of these phospholipases are efficient immunogens capable of inducing potent neutralizing antibodies with which they combine and react, for instance, the PLA2 subunit of the crotoxin(3,11). Thus, the PLA2 is one venom component endowed with quite well defined biochemical, biological and immunogenic properties. The present paper reports experiments showing that: a) the PLA2-dependent hemolysis in vitro induced by the venoms of Crotalidae and Elapidae snakes, can be used to quantify hemolytically the PLA2 present in these venoms; b) the curves obtained by plotting the degree of hemolysis (ordinate) versus the doses of venom (abscissa) are concave to the abscissa axis. We suggest that the hemolysis induced by PLA2 could be similar to that induced by the C system, therefore, obeying similar empirical equation; and c) antibodies present in the corresponding horse antivenoms specifically inhibit the PLA2-induced hemolysis.
MATERIAL AND METHODS
VENOMS: Venoms of Crotalus durissus terrificus (LD50=1,60 µg), Bothrops jararaca (LD50=38.38µg), Naja naja, Naja nigricollis, Naja melanoleuca, Micrurus ibiboboca, Micrurus frontalis (LD50=8.82 µg) and of Micrurus spixii were dissolved in 0.15M NaCl at the concentration of 2.0mg/ml and stored in aliquots at -20°C. Starting solutions at 12g/ml also in 0.15M NaCl were prepared before use.
ANTISERA: We used five anti-Crotalus durissus terrificus (L.-9210236, ED50=2.35; L.-9208187, ED50=2.22; L.-9209200, ED50=2.40; L.-9211267, ED50=1.99; L.-9211268, ED50=2.54), eight anti-Bothrops (L.-9212282, ED50=7.37; L.-9204072, ED50=6.71; L.-9209210, ED50=8.46; L.-9207171, ED50=7.12; L.-9212287, ED50=5.93; L.-9212291, ED50=6.56; L.-9210238, ED50=7.47; L.-9211246, ED50=7.10), two anti-Micrurus (L.-63, ED50=24,12, L.-64, ED50=44.08), and seven anti-tetanic sera, (L.-9111259\T.314\, Titer=1200UI/ml; L.-9211261\T.323\, Titer=>1400UI/ml; L.-9103063,\T.308\, Titer=110UI/ml; L.-911256\T.313\, Titer=1400UI/ml; L.-9303038, \T.325\Titer=140UI/ml;L. 9205104\T.318\ Titer= 1400Ui/ml; L.-9203057\ T.317\Titer=> 1400UI/ml). These antisera were prepared using hyperimmunized horses with the respective venoms, as described by Dias da Silva(2). All antisera were diluted 1:40 in 0.15M NaCl before use. The ED50 of the antivenoms were determined by the method of Finney(4). A lecithin (Merck Sharp Dome, USA) solution at 42µg/ml of saline containing 5µM CaCl2 was prepared.
SHEEP RED BLOOD CELLS (SRBC): The SRBC were washed five times with Triethanolamine-saline buffer pH 7.4 (TBS) and standardized to 1.5 x10 cell/ml.
DETERMINATION OF INDIRECT HEMOLYTIC ACTIVITY: PLA2-dependent hemolysis was assayed according to Tambourgi et al.(17). Briefly, increasing amounts of venoms starting from 120ng and increasing to 3000ng in a volume of 100 µl were added to a series of test tubes. To 100 µl of venom solution, 100 µl of lecithin solution in saline plus 0,01 M CaCl2, and 200 µl SRBC at 1.5 x 10 in TBS were added to each tube. Three control tubes each containing 200µl of TBS (blank), TBS plus lecithin (lecithin control) or 200µl of distilled water (100% lysis) were always included. After incubation for 1 h at 37°C, the reaction was stopped by adding 2.0 ml of cold saline to all tubes, except the 100% lysis tube to which 2.0 ml of water was added. The tubes were centrifuged for 10 min, the hemoglobin released in the supernatants was determined spectrophotometrically at 412 nm and the percentage of hemolysis was calculated. As the hemolytic curve obtained by plotting the percentage of hemolysis versus the doses of venom was concave to the abscissa axis, and following the von Krogh equation derived from the hemolysis induced by the C system, this equation was used to titrate the active PLA2. The presumptive of hemolytically active sites (Z) of phosphatidyl-choline molecules released from the lecithin by PLA2 can be represented by Z= - Ln (1-Y), the negative natural logarithm of the number of non-lysed erythrocytes, since Y is equal to the degree of lysis. For 62.3% hemolysis, Z=1, which corresponds to one hemolytically active site per erythrocyte (14). For the neutralization experiments with antivenoms, each venom solution previously titrated for its PLA2 content, was adjusted to contain 50 Z per milliliter.
NEUTRALIZATION OF THE PLA2-DEPENDENT HEMOLYSIS ACTIVITIES PRESENT IN SNAKE VENOMS: One hundred microliter samples of the venom solutions containing the amount of venom able to produce hemolysis correspondent to 1 to 5 Z were mixed with equal volumes of different dilutions of the antivenoms. The mixtures were allowed to stand at room temperature for 1h. Then, the samples were centrifuged for 30 min at 3,000rpm to remove the immunoprecipitates. The active PLA2 that remained in each experimental sample was assayed for its capacity to promote indirect SRBC hemolysis, and the residual number of Z was calculated as described above. Control samples containing venom (1 to 5Z) and the serum sample (lowest dilution) alone were always included. The necessary antibody dose to inhibit the hemolysis in each assay by the amount of venom used was chosen arbitrarily as the neutralization unit, which was referred one ED-100%.
DETERMINATION OF EFFECTIVE DOSE - 100% (ED-100): Using predetermined LD50 (B. jararaca, 38.38µg; C. durissus terrificus, 1.60µg; M. frontalis, 8.8µg), a range of doses of each venom was analyzed. For neutralization studies, venom and antivenoms were mixed together in saline and incubated for 1 h at room temperature before injection into groups of 4 or 5 mice. Control mice received an equivalent venom dose and control antivenom in saline, also incubated for 1 hour at room temperature. The mice were injected intraperitoneally with 100 to 1000 µl of solution. The animals were observed for 24h following the injections, and the results analyzed by the probit test.
PLA2-DEPENDENT LYSIS ACTIVITY PRESENT IN SNAKE VENOMS: In Figure 1 we represent one curve obtained after preliminary trials to determine the adequate dose range of each venom for optimal hemolysis under the standard assay conditions. The curves representing each venom are concave to the abscissa axis as in C-mediated lysis. Table 1 shows the number of Z per mg of the venom used. It is clear from these results that the analyzed venoms can be divided into two groups in regard to the PLA2-dependent hemolysis activity: the venoms of the genera Bothrops, Crotalus and Micrurus were the less active containing around 1 x 10 to 10 Z/mg of venom, while the venoms of Naja contained over a million Z/mg of venom.
TABLE 1. Determination of the number of effective molecules (Z) of lysophosphatidyl-choline generated from lecithin by some snake venoms.
NEUTRALIZATION OF THE PLA2-DEPENDENT HEMOLYSIS BY HYPERIMMUNE HORSE ANTIVENOMS: The ability of the horse hyperimmune antivenom to block the PLA2-dependent hemolysis was measured by preincubating constant amounts of the venoms at room temperature with a volume of the serum serially diluted and by measuring the remaining active PLA2 activity as described above. Typical results obtained for the PLA2-dependent hemolysis neutralization by horse hyperimmune antivenoms are shown in Figure 2. Antivenoms against Bothrops, Crotalus and Micrurus snake venoms specifically inhibited the PLA2-induced hemolysis by the corresponding venoms (Figure 2, Figure 3 and Figure 4). Antivenoms against Micrurus snake venoms were also able to inhibit Naja naja venom (Figure 3). Unspecific inhibitions were not observed either by the unspecific or by irrelevant antisera (Figure 5). Table 2 and Table 3 show the calculated numbers of ED-100 units per each antivenom analyzed.
TABLE 2. Comparison between the in vivo neutralization of lethality and in vitro inhibition of hemolysis producing activities of snake venoms by anti-bothropic and anti-crotalic sera. Heterologous venoms: a: N. melanoleuca; b: N. nigricolis; c: N. naja; d: M. spixii; e: M. ibiboboca.
TABLE 3. In vivo neutralization of lethality and in vitro inhibition of hemolysis producing activities of elapidic venoms (b: N. nigricolis; c: N.naja; d: M.spixii; e: M.ibiboboca) by anti-elapidic sera. The anti-tetanic sera were assayed with B. jararaca(f) and C. durissus terrificus(g) venoms as a negative control.
The PLA2-mediated hemolysis of SRBC can mimic in vitro the intravascular hemolysis observed in envenomation by animal venoms. In this paper, we showed that the lysis of SRBC induced by snake venoms in the presence of an excess of lecithyn and Ca ions followed a curve similar in shape to that induced by the C system, thereby, following the von Krogh equation. Thus, this equation could be used to calculate the presumptive number of lysophosphadyl-choline active molecules generated from the extracellular lecithin source and, indirectly, the effective number of lysophosphatidyl-choline molecules present in the venoms. Upon using this method, the number of lysolecithin molecules generated by the PLA2 present in seven snake venoms was shown to vary enormously, B. jararaca (1.0 x 10 molecules) and N. melanoleuca (39.9 x 10 ) standing on the lowest and highest extremes, respectively.
Upon knowing the presumptive number of lysophosphatidyl-choline active molecules generated by standard preparations of some snake venoms, a simple protocol to titrate antibodies against PLA2 present in these venoms was delineated. After preincubation of several dilutions of the antivenoms with a fixed amount of the corresponding venom, previously determined to generate 1 to 5 effective molecules of lysophosphatidyl-choline, the residual unblocked PLA2 molecules were determined by SRBC assay. The antibody titer necessary to neutralize the PLA2 responsible for the release of lysophosphatidyl-choline molecules was used to determine the neutralization unit and referred empirically as effective dose 100% units (ED-100).
The antibodies present in the horse antivenoms were capable of specifically neutralizing the PLA2 present in the corresponding venoms in a dose-effect-related fashion. No cross-reactivity was detected among antivenoms and non-corresponding venoms. A horse antiserum against tetanus toxin digested by pepsin and processed as the antivenoms was also unable to interfere with the venom PLA2 activity. The elapidic antivenom, however, besides being effective in the neutralization of the venoms of M. ibiboboca and M. spiixi, cross-reacted with the venom of N. naja, but not with the venom of N. nigricollis.
We hypothesize that antibodies against PLA2 present in the antivenoms can inhibit the enzyme by combining with epitopes surrounding the active site, thereby, preventing the enzyme appropriate interaction with the substrate. In this study, antibodies against the enzyme active site are not apparently involved in the antibody-PLA2 interaction, since lack of cross-reactivity was the rule. It has already been described that PLA2 of different snake venoms are recognized by heterologous anti-sera by ELISA and Western Blotting assays (13). This fact suggests that besides the molecular homology among these phospholipases, there is a heterogeneity among the enzymatic sites of PLA2 molecules or adjacent epitopes. Thus, antibodies produced against one species of venom can bind to heterologous venoms, but the binding does not affect the heterogenic region next to the enzymatic site, therefore, does not affect the enzymatic activity. In contrast, inhibition of the PLA2 present in N. naja venom by antivenom against Micrurus venoms can not be explained by zoological proximity among snakes of the two genera, since the PLA2 present in the other species tested such as N. melanoleuca were not inhibited. The higher PLA2 activity of N. melanoleuca venom can not account for the indirect hemolysis assay of the venoms because the latter were equalized to give 1 to 5 Z in the mixture.
The number of ED100 units per each antivenom reflects the anti-PLA2 antibody contents in the antivenoms. The high specificity and correlation degree with the neutralization of lethality indicate the use of the in vitro inhibition of the PLA2-mediated hemolysis as a substitute for the in vivo neutralization method. The in vitro methods are usually more sensitive, accurate and feasible besides being cheaper than the methods using animals. Furthermore, the use of in vitro methods in place of experimental animals meets the recommendations of the ethical principles on the use of animals in basic and in applied research. Therefore, the inhibition of PLA2-mediated hemolysis can be used to titrate antivenoms during the hyperimmunization of animals and the preparation of antisera.
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