Adjuvants--a classification and review of their modes of action.

Since early this century, various substances have been added to vaccines and certain formulations have been devised in an attempt to render vaccines more effective. Despite a plethora of options, only aluminium salts have gained acceptance as human vaccine adjuvants and even veterinary vaccines are largely dependent upon the use of aluminium salts. Currently, many new vaccines are under development and there is a desire to simplify vaccination schedules both by increasing the number of components per vaccine and decreasing the number of doses required for a vaccine course. New, more effective adjuvants will be required to achieve this.

An enzyme immunoassay for isotyping mouse monoclonal antibodies.

A rapid, simply performed and relatively inexpensive enzyme immunoassay for isotyping mouse monoclonal antibodies is described, based on the urease/urea system. Because of the high sensitivity (less than 0.1 microgram/ml of immunoglobulin can be detected in cell culture medium) no treatment of the hybridoma supernatant sample is required prior to assay, and the isotype of a mouse immunoglobulin can be determined in about thirty minutes.

Immunological stability of an elapid venom, Tropidechis carinatus, and its relevance to the clinical detection of snake venom.

Using immunological techniques, cases of human snake-bite can be proven and individual snake species identified. A series of experiments is described, to test the antigenic stability and physical properties of an Elapid venom, venom properties which will have implications in field use, transport, or the laboratory procedures relating to the immunological characterization of Australian snake venoms. Using venom from the Elapid, Tropidechis carinatus, we have demonstrated significant immunological stability at a temperature of 37 degrees for 48 h at least, and for 24 h at least when incubated continuously with skin, muscle, and fat homogenates; the venom is stable at 56 degrees for 2 h at least. No loss of venom occurs over the pH range 7-10; at pH ranges less than 4 and at ranges greater than 10 only 30% of venom is detected after 30 min incubation. Sonication (10 min at 100 watts) does not destroy immunological properties of the venom; 10 freeze-thaw cycles result in some 9% loss of immunologically detectable activity. Venom is adsorbed significantly on to dacron swabs, only 13% of the applied does being detected after 48 h incubation at 28 degrees. Sixty-seven percent of applied venom can be recovered from the skin of human volunteers 6 h after a simulated bite. The venom of this Elapid is antigenically robust. With the currently available sensitive assays, venom from human skin should still be detected in material kept without special preparation under field conditions for up to 2 days at least.

Further studies on the mass of venom injected by Elapid snakes.

Further experimental studies to determine the mass of venom injected by medically-significant Australian elapids are reported. The use of a modified enzyme immunoassay technique to measure venom injected during snake bite is presented. The feeding biting pattern of the Australian eastern brown snake (Pseudonaja textilis) is described. Using data from ten different snakes of this species, it is established that the mass of venom delivered in a first-bite is 4.69 +/- 0.85 mg (mean +/- S.E.) and a mean of 91% of the delivered venom is injected s.c. or into deeper tissues in a first-bite. For this species, the mass of venom delivered sequentially in a bite sequence falls to 1.32 +/- 0.94 mg in the third bite in such a sequence. For the Australian rough-scaled snake (Tropidechis carinatus), the mass of venom delivered in a first feeding bite is 6.15 +/- 2.23 mg, falling to a minimum of 1.92 +/- 0.61 mg in the third bite of a sequence. for the Australian death adder (Acanthophis antarcticus) the mass of venom delivered in a feeding bite is 41.95 +/- 16.13 mg for a first bite. Biting data is also presented for three species of the genus Pseudechis (the Australian mulga (king brown) and black snakes.

Monoclonal hybridoma antibodies against human IgE and their use in a rapid and sensitive enzyme immunoassay for the semiquantitative assessment of total IgE levels in human blood.

A simple semiquantitative enzyme immunoassay (EIA) for the rapid estimation of IgE levels in specimens of human blood, plasma or serum is described. The test requires little labour input and does not require highly trained personnel or instrumentation. By using two monoclonal antibodies of different anti-IgE specificities it is possible, with a single incubation of 20 min at ambient temperature, to detect elevated IgE levels (greater than or equal to 333 IU/ml) within a total test time of 25 min, and low levels of IgE (less than or equal to 10 IU/ml) within 35 min. For diagnosis of elevated/normal IgE levels only, a single incubation of 10 min. at ambient temperature may be used with a total test time of less than 20 min. The EIA system utilizes glass capillary tubes and urease-labelled antibodies, a system that has proven satisfactory in other applications.

The mass of venom injected by two elapidae: the taipan (Oxyuranus scutellatus) and the Australian tiger snake (Notechis scutatus).

Using an enzyme immunoassay technique, a new method for measuring, in vivo, the mass of venom injected during snake bite, is presented. The venom injected into mice (as prey) and the venom left on the skin surface during bites by the two Australian Elapidae, the Taipan (Oxyuranus scutellatus) and the Tiger Snake (Notechus scutatus) has been measured. Venom delivery patterns vary significantly between these two species. In the case of the Tiger Snake (a total of 45 bites studied) the mean mass of venom injected in a first bite was 12.7 mg (S.E. 3.4 mg, median 8.1 mg); an average mass of 0.8 mg (S.E. 0.4 mg, median 0.17 mg) was left on the skin surface. A second bite delivered by the same snake yielded a mean venom mass only 27% of the first. In the case of the Taipan (a total of 24 bites) the mean venom mass injected in the first bite was 20.8 mg (S.E. 6.4 mg); with an average of 0.9 mg (S.E. 0.5 mg) left on the skin surface. In contrast to the situation observed with Tiger Snakes, second and third bites delivered in a rapid sequence yielded increasing masses of venom. The mean delivered in the third of a sequence of three bites was 48.8 mg (S.E. 23.8 mg). The ranges of venom mass, by species and by the sequence number of the bite, are also presented. In 66 of the 69 experimental bites studied in this report, venom could be easily detected, the species identified, and the absolute mass of venom measured.

The estimation of bee venom specific human IgG.

Bee venom specific human IgG was measured by solid phase sandwich radioimmunoassay (RIA) using bee venom adsorbed to polystyrene tubes. RIA results were found to correlate with those obtained in an enzyme immunoassay (EIA) for bee venom specific IgG. The RIA did not suffer from the high background often associated with other solid phase radioimmunoassays for human IgG. Furthermore, both the RIA and EIA were simple to perform, inexpensive and highly reproducible.

Early management of bites by the eastern diamondback rattlesnake (Crotalus adamanteus): studies in monkeys (Macaca fascicularis).

Monkeys were injected subcutaneously with 6 mg of Crotalus adamanteus venom and a solid phase radioimmunoassay was used to measure levels of venom in plasma and urine. When no attempt was made to retard venom movement from the site of injection, plasma levels as high as 1,300 ng/ml occurred within 15 min of injection and progressive swelling developed in the injected limb. When first aid was employed (firm pressure to the injection site and immobilization of limb with a splint), plasma levels remained very low until cessation of first aid. No swelling of the injected limb occurred while the first aid measures were in position, and animals which received first aid an antivenom fared much better than did those which received antivenom alone. The best result was obtained when antivenom was infused prior to removal of the pressure bandages and splint. This first aid procedure is effective in delaying venom movement, and its simplicity and safety suggest it should be considered for use in cases of human envenoming by C. adamanteus.

Trypsin fails as Australian snake bite cure.

Trypsin has been claimed a new and effective treatment for venomous snake bite. We found that significant inactivation of snake venom lethal potency occurred in vitro when trypsin was incubated with venom and subsequently injected into mice. Premixing of tiger snake venom (TSV) and trypsin just before injection did not significantly increase the survival rate of mice over that of controls injected with TSV alone. Trypsin injected 10 to 30 minutes after TSV injection did not increase the survival rate of mice compared with controls. Specific antivenom was effective as an antidote when there was a 10 minute delay after venom injection. There was varying susceptibility of different venoms to trypsin inactivation in vivo.

Clinical laboratory: enzyme immunoassay for the rapid clinical identification of snake venom.

The treatment of snakebite could be simplified if the identity of the offending snake was more frequently known. A positive identification, which allows the use of a specific monovalent antivenom, probably occurs in less than 20% of cases. Recently published methods of venom detection (RIA and ELISA) take at least three hours to complete. We have developed a sandwich enzyme immunoassay (EIA) which is capable of detecting 0.5 ng of crude snake venom in about 90 minutes or 2 ng of crude venom in about 30 minutes. This substantial reduction in incubation times, while still retaining the sensitivity required, was due to the use of protein A purified rabbit IgG antivenom from hyperimmune serum and the enzyme horseradish peroxidase (HRPO). A rapid identification of the offending snake by this method may reduce the use of large-volume polyvent antivenoms, thus avoiding the clinical and economic disadvantages of such preparations. Other advantages would be an increased understanding of the clinical syndrome produced by the individual species of snake, and accumulation of data about the incidence of envenoming attributed to specific snakes.

Rationalisation of first-aid measures for elapid snakebite.

The plasma of monkeys envenomated with tiger snake (Notechis scutatus) venom was monitored by radioimmunoassay for both crude venom and a neurotoxin. When the injected limb was immobilised and a pressure of 55 mm Hg applied to the injection site, only very low levels of circulating venom or neurotoxin were detectable. In practical terms, venom movement can be effectively delayed for long periods by the application of a firm crepe bandage to the length of the bitten limb combined with immobilisation by a splint. Pressure alone or immobilisation alone did not delay venom movement.