First clinical experiences with specific sheep Fab fragments in snake bite. Report of a multicentre study of Vipera berus envenoming.

OBJECTIVES: To evaluate the efficacy and safety of specific, ovine Fab fragments in the treatment of envenoming by the common adder, Vipera berus. DESIGN: Open study with historical controls. SETTING: Multicentre study involving patients (n = 30) with V. berus envenoming, treated in 18 Swedish hospitals during 1991-94. MAIN OUTCOME MEASURES: Initial symptoms, clinical course after treatment, duration of hospital stay and adverse effects of the antivenom were evaluated. Two earlier studied patient groups, given either equine F(ab)2 antivenom (n = 30) or no antivenom (n = 16), were used as controls. RESULTS: Specific ovine Fab fragments influenced favourably the acute symptomatology as well as the long term clinical course. Acute symptoms such as hypotension, shock, vomiting, diarrhoea and CNS-depression resolved quickly. The incidence of extensive swelling involving the trunk and the length of hospital stay were both reduced significantly compared to nontreated patients (23 vs. 88% and 3.5 vs. 6 days). Also the incidence of anaemia was reduced (23 vs. 44%). These results were consistent with those obtained with equine F(ab')2 antivenom, but with ovine Fab there were no immediate anaphylactic reactions or serum sickness. CONCLUSION: Specific Fab fragments produced from sheep immunized with V. berus venom were safe and effective in counteracting the effects of V. berus bite in humans. These results justify further studies of this new treatment for snake envenoming.

Assessment of an ovine antivenom raised against venom from the desert black cobra (Walterinnesia aegyptia).

The desert black cobra (Walterinnesia aegyptia) is an elapid widely distributed throughout the deserts of Saudi Arabia and currently available antivenoms are ineffective in the treatment of its envenoming. Walterinnesia aegyptia venom was assessed for several of its physicochemical, enzymatic and biological characteristics. An antivenom was raised in sheep using a low-dose immunization schedule and digested with papain to provide Fab fragments. The antivenom neutralized all of the above enzymatic and biological activities and provided good protection in mice (ED50 0.25 g/kg), whereas the commercial polyspecific products showed only partial neutralization and did not protect mice.

Development and clinical application of immunoassays for European adder (Vipera berus berus) venom and antivenom.

An ovine affinity purified Fab antivenom was used in a clinical trial in Sweden to treat European adder (Vipera berus berus) envenoming. Immunoassays were developed to measure V. b. berus venom and antivenom concentrations in clinical samples to help assess the efficacy of treatment. A radioimmunoassay (RIA) was developed, optimized and validated to measure plasma levels of V. b. berus venom and compared with a conventional ELISA. Both showed a similar variation of zero binding in biological samples and the results obtained correlated closely. However, the ELISA was quicker and more sensitive (0.8 compared with 2 micrograms/litre). Before administration of antivenom, V. b. berus venom concentrations in plasma ranged from 10 to 53 micrograms/litre; 12 hr after the Fab infusion, no patient had measurable levels. However, two patients had low venom levels 24 hr after treatment. ELISA and RIA were also developed, optimized and used to measure concentrations of free Fab in plasma. There was a biexponential fall of Fab concentration with a fast distribution phase (t 1/2 = 0.9 hr) and a slower elimination phase (t 1/2 = 18 hr). The amount of Fab excreted in urine was low.

A comparison of ovine and equine antivenoms.

Commercial antivenoms produced in horses were compared with monospecific antivenoms raised in sheep against Crotalus durissus terrificus, Crotalus atrox, Crotalus adamanteus, Micrurus fulvius fulvius, Naja naja, Naja kaouthia, Echis ocellatus, Vipera lebetina deserti, Vipera berus berus and Vipera ammodytes ammodytes venom. Antibodies raised by immunizing sheep with C. d. terrificus venom were more effective than their equine counterparts in preventing lethal toxicity in mice (ED50), in inhibiting the venom's pharmacological effects (haemolysis, platelet aggregation and coagulation), and in neutralizing phospholipase A2 activity. Comparison of one ovine and three equine F(ab)2 products raised against V. a. ammodytes venom showed that all were at least 95% pure; that all protected mice; and that all contained antibody populations directed against most components of V. a. ammodytes and V. b. berus venoms. The ovine antivenoms generally contained a higher concentration of specific antibodies than the equine products. Finally, the ovine antivenoms raised against E. ocellatus, V. lebetina deserti, V. b. berus, M. f. fulvius and N. naja venoms provided better in vivo protection to mice than the equine antivenoms, but the equine antivenoms to N. kaouthia and C. atrox were more protective than the ovine product.

An indirect haemolytic assay for assessing antivenoms.

Dilutions of antivenom, venom, human erythrocytes and a phosphatidylcholine suspension, were incubated for 30 min at 37 degrees C. After centrifugation, the liberated haemoglobin was measured spectrophotometrically. The assay was used to assess an ovine antivenom against the venom from the South American rattlesnake, Crotalus durissus terrificus, and an equine Wyeth antivenin (Crotalidae, polyvalent). The ovine antivenom was more than five times as effective as the equine product. It also neutralized venoms from the Western diamondback rattlesnake, Crotalus atrox, and the fer-de-lance, Bothrops atrox. However, antivenoms raised against venoms from other Crotalus and Bothrops species provided little protection against the haemolytic activity of C. d. terrificus venom.

Comparison of three different activation methods for coupling antibodies to magnetisable cellulose particles.

The periodate and 1,1'-carbonyldiimidazole activation methods were compared with the cyanogen bromide procedure for coupling antibodies to magnetisable cellulose/iron oxide solid-phase particles. Fluoroimmunoassays for quinine, primaquine, normetanephrine and cannabinoids were employed to assess the binding properties of such coupled solid phases. The cyanogen bromide and 1,1'-carbonyldiimidazole methods gave similar products in most cases, while the specific binding capacity of periodate coupled particles was between two and five times lower. Nevertheless, comparable standard curves could be obtained with solid phase coupled by each method. The periodate and 1,1'-carbonyldiimidazole methods are acceptable alternatives, notably for laboratories lacking the facility to handle the toxic cyanogen bromide.

Development of a magnetisable solid-phase fluoroimmunoassay for primaquine and carboxyprimaquine.

Primaquine coupled to keyhole limpet hemocyanin was used as an immunogen to produce antiprimaquine antibodies in three sheep. The antisera obtained were characterised by the increase in fluorescence polarisation found upon binding to fluorescein-labelled primaquine prepared via same route. All sheep showed a good antibody response and one antiserum was coupled to magnetisable solid-phase particles to facilitate the separation of the antibody bound from free labelled antigen and the removal of interfering components which may be present in the sample. The fluoroimmunoassay requires addition of 100 microliters of standard or sample (urine or serum) to 100 microliters tracer (150 nmol/l) followed by 100 microliters of magnetisable solid-phase particles (12.5 g/l). After one hour incubation followed by the usual washing and eluting procedures, using a magnetic rack, the fluorescence of the supernatant was measured directly in a fluorimeter. Sodium salicylate was incorporated in the tracer solution to block the non-specific binding of tracer to the protein in serum samples. Cross-reactivity studies showed that the antibodies have high specificity for the 8-aminoquinoline nucleus but not to the 8-N-aminobutyl side chain. Thus carboxyprimaquine cross-reacted equally with primaquine and the assay can be used to measure their combined level. After extraction of primaquine from a basified sample with methylene chloride, the assay may be applied for the quantitation of either primaquine (in the organic phase) or its acidic metabolites including carboxyprimaquine (in the aqueous phase) separately. This approach was applied for the determination of total primaquine (primaquine and its metabolites) and extracted primaquine in urine samples following a single oral dose of 45 mg primaquine.(ABSTRACT TRUNCATED AT 250 WORDS)

Polarisation fluoroimmunoassay for quinine in serum and urine.

The development and validation of a polarisation fluoroimmunoassay for the antimalarial drug quinine is described. The assay is performed either by sequential addition of the reagents or by a single-reagent technique whereby the tracer and antibodies are premixed. Serum samples require pepsin digestion prior to assay while urine specimens are assayed directly. The reliability criteria of the assay are satisfactory and no cross-reaction is detected with quinidine (the optical isomer of quinine) or with common antimalarial drugs. The assay was applied to the measurement of quinine in urine specimens obtained from a single-dose pharmacokinetic study and the results correlated with those of the benzene extraction fluorescence method for quinine measurement.

Quinine directly determined in serum or urine by separation fluoroimmunoassay.

We developed and validated a magnetizable solid-phase fluoroimmunoassay for directly determining quinine in serum or urine. We prepared the immunogen by coupling quinine hemisuccinate to keyhole-limpet hemocyanine, using this to immunize three sheep, and coupling the antisera to magnetizable solid-phase particles to facilitate separation of bound antigen from interfering components of serum or urine. We tested the stability of two fluorescein-labeled derivatives of quinine--one prepared by direct conjugation of quinine to dichlorotriazenyl aminofluorescein, the other by conjugating quinine hemisuccinate to fluorescein thiocarbamyl ethylenediamine. The latter was unstable. Using the former label and an 30-min incubation, we saw no interference by quinidine (an enantiomer of quinine) or other antimalarial drugs at their therapeutic concentrations.