Neutralization of bee venom lethality by immune serum antibodies.

The lethal effects of Africanized honey bee venom depend on the absorption of venom delivered during simultaneous sting attacks by large numbers of bees. The hypothesis that antibodies to whole bee venom and bee venom components could neutralize the lethal effect of bee venom was tested. Antibodies from beekeepers and immunized rabbits were incubated with bee venom and neutralization was studied by survival of intravenously injected mice. Beekeeper serum antibodies were found effective in protecting mice challenged with whole venom, and serum from rabbits immunized with phospholipase A2 (PLA2) was effective in protection against lethal effects of PLA2. Serum antibodies from rabbits immunized with whole venom or melittin were ineffective in neutralizing whole venom in vivo and had low titers in a venom enzyme-linked immunosorbent assay. The results suggest the need for development of more effective methods for raising antitoxic antibodies to bee venom components in other animals as a means of developing an antiserum that would be effective for treatment of human victims of multiple bee stings.

Ovine desipramine antibody fragments reverse desipramine cardiovascular toxicity in the rat.

STUDY OBJECTIVE: To develop a model of severe desipramine cardiovascular toxicity and to determine whether partial neutralization of the antigen by desipramine-specific Fab antibody fragments ameliorates its cardiovascular effects. METHODS: We administered desipramine to rats until the QRS interval tripled in duration and mean arterial pressure (MAP) was less than 100 mm Hg. Animals were then assigned to one of six groups: (1) no treatment, (2) normal saline solution control treatment (.9% NaCl infusion equal to the volume of Fab infusion), (3) nonimmune Fab control treatment (infusion of Fab equal to that in the 9.6% neutralization treatment), (4) 9.6% desipramine Fab (infusion of ovine desipramine Fab equal to 9.6% of an equimolar neutralization), (5) 19.2% desipramine Fab, and (6) 30.0% desipramine Fab. RESULTS: QRS-interval duration, heart rate, and MAP were recorded for 60 minutes. Animals in groups 1 through 3 demonstrated slow and incomplete improvement. Animals in groups 4 through 6 showed improvement in QRS-interval duration and heart rate within 4 minutes (P<.05) compared with untreated animals. A dose-response relationship was evident; animals given the highest dose of desipramine-specific Fab showed the greatest improvement. CONCLUSION: Partial neutralization of desipramine by specific Fab fragments produces rapid improvement of QRS-interval duration and heart rate in a rat model of severe desipramine toxicity.

Comparison of a new ovine antigen binding fragment (Fab) antivenin for United States Crotalidae with the commercial antivenin for protection against venom-induced lethality in mice.

Snake venom poisoning is a medical emergency requiring immediate attention and the exercise of considerable judgment. Of the estimated 8,000 bites inflicted by venomous snakes in the United States each year, approximately 6,000 are treated with commercial antivenin. The only commercially available antivenin for North American Crotalidae envenomation is Antivenin (Crotalidae) Polyvalent (equine origin) (ACP; Wyeth Laboratories, Philadelphia, PA). A common complication is the high incidence of hypersensitivity reactions, occurring in more than 75% of patients treated with ACP. To minimize these side effects, a novel, affinity-purified, antigen binding fragment (Fab) antivenom (FabAV) for Crotalidae venom poisoning has been produced from the sera of sheep. The new product is Antivenin Polyvalent Crotalid (Ovine) Fab (Crotab; Therapeutic Antibodies, Inc., Nashville, TN). The current report compares the potencies in mice of FabAV and ACP against venom-induced lethality. The results indicate that FabAV is 3.1-9.6 times more potent than ACP for the prevention of lethality of the nine United States venoms tested. For one of the venoms, Crotalus viridis helleri, FabAV was efficacious while ACP was not.

Significance of Africanized bees for public health. A review.

Although massive sting attacks by Africanized bees are currently rare, this type of bee is now endemic in parts of Arizona and Texas, and will probably spread to other warm climate areas in the United States. Treatment of severe toxic reactions to multiple stings usually includes management of shock, hypoxia, and other effects of organ damage. New approaches to reduce blood levels of venom including production of a bee antivenom and hemodialysis require further study. Patients with a trivial allergy to single stings could be at risk from systemic anaphylaxis to multiple bee sting. Those who wish to remain in endemic areas in spite of histories of systemic anaphylaxis to bee stings should be treated with venom immunotherapy, possibly administered more intensively and for longer periods than currently recommended. Continued public education is needed to limit contact with aggressive Africanized bee colonies.

A molecular-based magnet test for malaria.

The molecular-based magnet test for malaria is shown to be more sensitive than the thin blood film test. The globally used thin blood film test is less sensitive because it uses preparation steps that result in the reduction of the absolute number of diagnostically pertinent erythrocytes. Several reports of diagnostic error with the thin film test and the thick film test have appeared in the literature. In marked contrast to the commonly accepted tests, the magnet test concurrently partitions and concentrates the infected erythrocytes present in the initial sample. The magnetic test permits a brief and sensitive microscopic-based enumeration of the malaria-infected erythrocytes in the enriched sample. Diagnostically pertinent hemozoin is simply identified through two of its specific molecular properties: paramagnetism and birefringence. The former property mediates the capture and enrichment of malaria-infected erythrocytes within the magnetic flux and the latter property manifests the characteristic birefringence demonstrated by polarized light.

Rate and quantity of delivery of venom from honeybee stings.

To determine the rate and completeness of delivery of venom from honeybee stings, European bees were collected at the entrance of a hive and studied with the use of two laboratory models. In one model bees were induced to sting the shaved skin of anesthetized rabbits. The stings were removed from the skin at various time intervals after autotomization, and residual venom was assayed with a hemolytic method. In the other model the bees were induced to sting preweighed filter paper disks, which were weighed again after removal of the sting at various intervals. Results of both experiments were in agreement, showing that at least 90% of the venom sac contents were delivered within 20 seconds and that venom delivery was complete within 1 minute. The data suggest that a bee sting must be removed within a few seconds after autotomization to prevent anaphylaxis in an allergic person. The extensive variation found in the amount of venom delivered at each time point may explain inconsistencies in relationships among reactions to field stings, sting challenge testing, venom skin tests and RAST.

Effects of constriction bands on rattlesnake venom absorption: a pharmacokinetic study.

STUDY OBJECTIVE: To determine whether the use of a constriction band alters systemic absorption of rattlesnake venom in pigs and whether constriction band use alters local swelling. DESIGN: Using a crossover design, five pigs were studied with and without the use of a constriction band. 125I-Labeled Western Diamondback rattlesnake (Crotalus atrox) venom was injected subcutaneously into one foreleg. The protocol was repeated using the opposite foreleg six days later. The constriction band was applied at the time of injection and removed four hours later. Plasma radioactivity and leg circumference were measured serially. RESULTS: Maximum plasma venom concentration and area under the venom concentration-time curve were compared in trials with and without constriction band. Within the initial four hours, application of a constriction band decreased maximum plasma venom concentration by 25% and area under the venom concentration-time curve by 33% (P less than .05). After the constriction band removal at four hours, maximum plasma venom concentration and the area under the venom concentration-time curve were not significantly different between groups. Application of a constriction band did not result in a statistically significant increase in maximum leg circumference as compared with trials without a constriction band. CONCLUSION: The use of a constriction band was effective in reducing venom absorption while it was in place (reduced area under the venom concentration-time curve and maximum plasma venom concentration in the cuffed group), and constriction band removal did not result in a significant increase in maximum plasma venom concentration. Leg swelling was not affected by constriction band use. Because constriction band use delayed venom absorption without causing increased swelling, it may prove to be a useful first aid measure in human beings.

Biochemical variability of venoms from individual European and Africanized honeybees (Apis mellifera).

To study biochemical differences between venom from individual honeybees, venom sacs from 103 European (EU) bees and 92 Africanized bees representing 12 different colonies were dissected, and the dry weight (DW) of venom from each bee was determined. Venom from each of these bees was studied with isoelectric focusing and functional assays for phospholipase A2 and melittin. Phospholipase concentrations in individual EU bee venoms varied between 1.8% and 27.4% (wt/wt). The melittin concentration in EU bee venom varied less and, on the average, was found to be much lower than previously reported. There was an eightfold to ninefold difference between lowest and highest venom sac DW contents, suggesting the possibility of highly variable venom delivery from bee stings. One EU bee contained greater than 300 micrograms of venom, three times the recommended maintenance dose for venom immunotherapy. Isoelectric focusing also demonstrated large differences between individual bees, with respect to major and minor components of their venoms. Africanized bees contained significantly less venom but more phospholipase than did EU bees. Bee venoms from different colonies differed in their DW content and in their concentrations of phospholipase and melittin. The results are relevant to the uncertainty of responses from sting challenges and field stings in allergic patients and massive stinging attacks on normal subjects.

Membrane active plasma factor in multiple sclerosis: characterization and isolation by recycling isoelectric focusing.

Recycling isoelectric focusing is a rapid, high resolution technique that has the capability of fractionating complex mixtures of proteins on a preparative scale on the basis of their isoelectric points (pIs). For this reason, it appeared to be an ideal tool to further characterize and isolate the surface active plasma component(s) which is abnormal in multiple sclerosis (MS). The normal control and the abnormal MS plasma components, or factors, proved to be stable under the conditions used in this technique, including deionization by electrodialysis, dialysis against distilled water, lyophilization and the presence of 3M urea and carrier distilled water, lyophilization and the presence of 3M urea and carrier ampholytes. The presence or absence of plasma factor activity was determined by incubating red blood cells in a test plasma, or plasma fraction, followed by the determination of the red blood cell electrophoretic mobility in the presence and absence of linoleic acid. Both the normal and MS factor had a pI of 4.0 +/- 0.1 under the conditions used. A high degree of purification was achieved and albumin was eliminated as a possible candidate for the factor(s).

Quantity, analysis, and lethality of European and Africanized honey bee venoms.

Venom from Africanized honey bees (derived mainly from Apis mellifera scutellata) was compared with venom from domestic, European bees by study of lethality, immunological cross-reactivity, venom yield, isoelectric focusing (IEF) patterns, and melittin titers. The LD50s of European and Africanized bee venom by iv injection in mice were similar. In venom neutralization experiments, Africanized bee venom was mixed with antibodies from a beekeeper exposed only to European bees and used to challenge mice. Survival times of mice given these mixtures were significantly prolonged, indicating that human serum antibodies to European bee venom neutralized the lethal effects of Africanized bee venom. Reservoirs from Africanized bees contained less venom than European bees (94 and 147 micrograms venom/bee, respectively) and Africanized bee venom had a lower melittin content. The IEF patterns of venom from individual European bees varied considerably, as did IEF patterns of individual Africanized bees. Pools of venom from 1,000 bees of each population of A. mellifera showed noticeable but less obvious electrophoretic differences. The findings suggest that multiple stinging, and not increased venom potency or delivery, is the cause of serious reactions from Africanized bee attacks.

Purification of the phospholipase D of Corynebacterium pseudotuberculosis by recycling isoelectric focusing.

Recycling isoelectric focusing was investigated as a method for purification of phospholipase D (PLD) from cultures of Corynebacterium pseudotuberculosis. Supernatant fluids from cultures of equine isolate 155 in brain-heart infusion broth were dialyzed against distilled water, concentrated by lyophilization, and fractionated by preparative isoelectric focusing in free solution in a pH 3 to 13 gradient with 6M urea. Protein concentration, pH, and PLD activity of the 10 resulting fractions were determined. Two PLD activity assays were used: release of 14C choline from labeled sphingomyelin and synergistic hemolytic activity with Rhodococcus equi factors. Enzyme activity focused in 2 fractions at pH 8.5 to 9.8. The synergistic hemolytic assay was simple and rapid for detecting PLD in partially purified fractions. Electrophoretic examination of the fraction containing the highest concentration of PLD activity revealed protein bands at 14, 21, and 31.7 kD Mr, suggesting purification to near-homogeneity. Proteins from the 31.7-kD band were labeled by antibodies in serum from a goat with chronic C pseudotuberculosis infection.

Purification of lentil lectins using preparative electrophoresis.

Electrophoresis presents an interesting alternative to chromatography for the purification of biological compounds. To document the performance of three preparative electrophoresis apparatus currently available, they were applied to the purification of lectins from lentil seeds which contain two isolectins usually purified by chromatography. Purification by electrophoresis consists of first isolating a mixture of the two isolectins and then separating them. For the first step, either of two free-flow electrophoresis apparatus were employed: the Elphor VaP 22, using field step electrophoresis and the Biostream using zone electrophoresis. To optimize the process, the Biostream was modified to a recycling mode. This required repositioning one dialysis membrane which separates an electrode from the separation chamber. This allowed the lentil extract to be desalted by electrodialysis directly in the apparatus prior to fractionation. A high concentration of lectins was collected at the cathode and acidic proteins were collected at the anode. The bulk of the extract was recycled until the whole volume was processed. In a second step the isolectins were separated by recycling isoelectric focusing in the recycling isoelectric focusing apparatus. The present work clearly demonstrates that electrophoretic methods provide lectins with higher purity than chromatographically purified commercial products.

Isolation of monoclonal antibodies to phencyclidine from ascites fluid by preparative isoelectric focusing in the Rotofor.

A monoclonal antibody to phencyclidine was developed, produced in mouse ascites fluid, and purified. The purification used only preparative-scale isoelectric focusing in the Rotofor and dialysis. In 4 h, 25% (4 mg) of the antibody from 10 ml of ascites fluid was purified to homogeneity while 63% of the total antibody was recovered.

Isolation by preparative isoelectric focusing of a direct acting fibrinolytic enzyme from the venom of Agkistrodon contortrix contortrix (southern copperhead).

Fibrolase, a blood clot-lysing enzyme, was isolated from the venom of the snake Agkistrodon contortrix contortrix using preparative scale isoelectric focusing in the recycling isoelectric focusing (RIEF) apparatus. Two sequential purifications, beginning with 1.0 g of whole, dried venom, were employed. A pH 6-8 range gradient effected the first separation. While 100% of the enzyme was recovered in three fractions, 43% (one fraction) had 70% purity. The second run was a refractionation of three, pooled fractions from the first run, in a 0.7 pH range gradient. Of the fibrolase in the venom, 63% was recovered in four fractions. One of these represented 29% of venom fibrolase, with 97% purity. Gel filtration chromatography removed most of the remaining, higher molecular weight contaminants of the RIEF-purified enzyme.

An isoelectric focusing study of seasonal variation in rattlesnake venom proteins.

Adult specimens of seven southern Pacific rattlesnakes (Crotalus viridis helleri), four northern black-tailed rattlesnakes (Crotalus molossus molossus), and six western diamondback rattlesnakes (Crotalus atrox) were housed under controlled light and temperature and milked of venom monthly for 20 months. Ambient conditions were modelled to simulate seasonal change. Weighed amounts of lyophilized venom from each snake were compared chronologically for variation in isoelectric focusing patterns, using natural and immobilized gradients. No variation in patterns was evident over this time period for any individual snake. However, intraspecific differences were obvious in the venom samples. The pattern seems indicative of a species, however, concentration of various protein constituents seems individual and genetically "fingerprinted'. Unlike other physiological functions that demonstrate cyclicity in response to temperature and photoperiod, concentration ratios of venom components appear to be constant regardless of external cues. These findings may further emphasize the medical importance of treating snakebite victims symptomatically as individuals. A variation exists in the components of venoms of any given species, as well as in the physiological sensitivities of humans to a venom.

Characterization of synthetic carrier ampholytes by repetitive scanning of the electric field during focusing.

This paper reports the utilization of a potential gradient array detector for monitoring the dynamics of the electric field during isoelectric focusing. Transient and steady state electric field profiles are presented for synthetic carrier ampholyte mixtures with a wide (approximately 3-10) pH range. Two available commercial products (Ampholine and Pharmalyte) and a laboratory synthesized mixture (PEHA ampholytes) are compared. The formation of conductivity gaps and their migration toward the cathode in extended experiments (cathodic drift) can be visualized with this system.

Preparation of a new antivenin by affinity chromatography.

Polyacrylamide gel affinity chromatography was employed to isolate and purify antibodies to the antigens of the venoms of four rattlesnakes. The antivenins were studied for their neutralizing properties on a number of pharmacologic preparations. It was found that the purified antibodies (IgG) were more efficacious than the commercially prepared antivenin in neutralizing the lethal, cytolytic, hemorrhagic, platelet aggregating, and other deleterious effects of the venoms. In addition, the purified antibodies gave no evidence of producing anaphylaxis or anaphylactoid reactions in animals sensitized to horse serum. The proposed technique is also more simple than current production methods, is time-saving, and less expensive.

Effects of preparatory procedures on the venom from a rattlesnake (Crotalus molossus molossus), as determined by isoelectric focusing.

A study was made on the venom of a single specimen of the rattlesnake Crotalus molossus molossus, utilizing isoelectric focusing in polyacrylamide gels, to determine what effects various commonly employed preparative procedures might have on the chemical properties of the venom. The procedures included storage at several temperatures, freezing and thawing, evaporation, desiccation and lyophilization. Results indicated that the numbers and relative positions of the protein bands were constant with each procedure. It was concluded that the various procedures did not cause enzymatic degradation or major shifts in the pI of the bands. The significance of the findings is discussed.

Properties of narrow-range ampholytes isolated from wide-range ampholyte preparations.

A simple procedure for obtaining useful narrow-pH-range ampholytes from inexpensive laboratory-synthesized ampholytes by preparative isoelectric focusing in Pevikon is described. The narrow-range ampholytes prepared in this way are comparable to commercial ampholyte preparations as judged by conductivity, buffer capacity, pH gradient formation, and resolving power. These inexpensive narrow-range ampholytes are particularly well suited to preparative isoelectric focusing applications requiring large quantities of ampholytes.