In vitro assay of biological and chemical toxins using antibodies against lethal toxin neutralizing factor

Lethal Toxin Neutralizing Factor (N-LTNF), MW 63.0 kDa, was isolated from opossum serum. After trypsin digestion, the active domain of N-LTNF was isolated and sequenced. The synthetic peptide consisting of ten amino acids was designated as LT-10. N-LTNF and LT-10 inhibited the lethality of animal, plant and bacteria toxins when tested on mice non-immunologically. The antibodies against N-LTNF and LT-10 only reacted immunologically with toxins and not with non-toxic substances. Anti-LTNF and anti-LT-10 reacted immunologically by ELISA test with toxins that were not detected by mouse test, such as cholera toxin and digoxin. Anti-LTNF and anti-LT-10 failed to react immunologically with non-toxic substances, such as nerve growth factor and collagen. Currently, mouse bioassay is in use for toxin detection and assay. Binding affinity of IgG from anti-LT-10 showed a linear relationship with mouse bioassay by ELISA detection limit to some toxins only. This may be due to the fact that anti-LTNF and anti-LT-10 detected the toxins that were not lethal to mouse. Thus, anti-LTNF and anti-LT-10 can be useful in assaying toxins as an alternative to mouse bioassay.

Sub-lethal injection of honeybee venom decreased the levels of endogenously present substances in organs of mice

Pharmacological substances such as adenosine deaminase (ADA), collagen, histamine, IgE, myoglobin, and nerve growth factor (NGF) are endogenously present in animals. Research from this laboratory reported decreased levels of ADA, histamine, IgE, and NGF in organs of mice injected with sub-lethal doses of cobra venom. The goal of this research is to observe the levels of ADA, collagen, histamine, IgE, myoglobin, and NGF in certain organs of mice injected with venom from the bee Apis mellifera. Adult Balb/c female mice IM injected with half lethal dose of bee venom were sacrificed after 2 and 8 hours for removal of organs. The homogenates of the organs were assayed by ELISA for ADA, collagen, histamine, IgE, myoglobin, and NGF using respective antisera. Organs from mice injected with PBS were used as controls. It was observed that there were decreased levels of ADA, collagen, histamine, IgE, myoglobin, and NGF in certain organs after 2 h and tremendous decrease after 8 h. This is the first report showing the pharmacokinetics of ADA, collagen, histamine, IgE, myoglobin, and NGF as consequence of honeybee envenomation.

Decreased levels of nerve growth factor in organs of mice as a consequence of sub-lethal injection of cobra venom.

Nerve growth factor (NGF) is endogenously present in salivary glands of mice and sex organs of various animals. This research reports the presence of NGF in almost all major organs of mice at varying concentrations. The research further reports that intramuscular injection of a sub-lethal dose of Naja kaouthia venom lowered the levels of NGF in the organs of mice. Adult Balb/C male mice were injected with a half lethal dose of cobra venom. The mice were sacrificed for organs at 2, 8, and 24 hours post injection. Organs were homogenized, centrifuged, and the supernatants were assayed for NGF using anti-NGF by immunological tests enzyme-linked immunosorbent assay (ELISA). The organs of the mice injected with PBS served as controls. Major decrease in the levels of NGF was observed 2 hours after venom injection, and tremendous decrease of NGF was observed in organs of mice 24 hours post injection. The most lowering for NGF was observed in brain, heart, liver, salivary gland, and testis. This is a first-hand investigation showing the pharmacokinetics of NGF in organs of mice as an effect of envenomation.

Isolation and characterization of nerve growth factor (NGF) excreted from cultured eukaryotic cells.

Since the discovery in 1954, NGF has been isolated from snake venoms and various tissues and organs of different animals. Recently, Lipps (2000b) reported the isolation of NGF from the human body fluids, saliva, serum, and urine. This investigation reports the isolation of NGF excreted by various types of cells from diverse origin, in serum free medium, proving that its presence is not restricted to neural cells. The established cell lines used were Chang's liver and neuroblastoma of human origin, Vero monkey origin, PC12 rat, and SP/2 mouse origin. The fully grown monolayers of the cells were maintained in serum free medium for 48 hours to excrete NGF in the medium and the cell free medium was concentrated. NGF from cell free concentrated medium for each cell line was isolated by high pressure liquid chromatography (HPLC) and was identified as described by Lipps (1998). The HPLC profiles for cell free concentrate from different types were observed to be similar. The NGF fraction was eluted last in the neutral pH (Fig. 1). The identified fraction for NGF from each cell line was further purified, which resolved into a single peak. The purified NGF was used to study the biological and immunological properties. The biological activities of NGFs from cell free medium were minuscule in comparison to the cobra venom derived NGF. The molecular weights of NGFs from cell free medium for all cell lines were identical, 36.0 kDa. Anti-human NGF reacted strongly immunologically with NGFs from human origin cells and poorly with rat and mouse. PC12 NGF antibody reacted immunologically only with PC12 NGF.

Production of polyclonal antibodies in mice against cobratoxin, botulinum toxin and ricin without altering their toxicity or use of adjuvant.

Purified venom components, botulinum toxin and ricin have been successfully used as immunogenes, after converting to toxoids and using adjuvant for production of polyclonal antibodies in animals. This communication reports that polyclonal antibodies specific to cobratoxin, botulinum toxin and ricin were generated in Balb/C mice. The toxins were used for immunization without adjuvant and without altering their toxicity or converting them to toxoids. Initially, lethal dose for botulinum toxin, cobratoxin and ricin were determined in mice and found to be 1 microg, 4 microg and 2 microg, respectively. For the production of antibodies mice were injected with half lethal dose of the toxins in natural form four times, two weeks apart. The potency of antitoxins was assayed by enzyme-linked immunosorbent assay. High titer antibodies were generated by botulinum toxin, cobratoxin and ricin after three injections consisting of half mouse lethal dose. Such minute amounts of botulinum toxin, cobratoxin and ricin in their natural form were able to produce high titer antibodies, perhaps because these toxins may fall in the category of super-antigens.

Isolation of nerve growth factor (NGF) from human body fluids; saliva, serum and urine: comparison between cobra venom and cobra serum NGF.

Several investigators have isolated nerve growth factor (NGF) from various tissues and organs of different animals. There is no published documentation about NGF from body fluids, such as blood serum, saliva, and urine. Contrary to the unsuccessful attempts to detect or isolate NGF in serum in the past, this investigation reports the isolation of NGF from human serum, saliva, and urine. It further reports the comparison of properties between NGFs derived from cobra venom and cobra serum. NGF from serum, saliva, and urine was isolated by high pressure liquid chromatography (HPLC) and was identified as described by Lipps (1998). The identified fractions of NGF were further purified to study the biological and immunological properties. The biological activities of NGFs from human body fluids and cobra serum on PC12 cells were miniscule in comparison to the cobra venom derived NGF. The molecular weights of NGFs from human serum, saliva, and urine were identical, 36.0 kDa. However, the molecular weights of cobra serum and cobra venom NGFs were different, 55.0 kDa and 13.5 kDa, respectively. NGF level is age dependent and varies under different conditions. Using anti-human NGF, diagnostic tests can be developed for neurological disorders. This investigation also emphasizes the replacement of invasive blood collection for serum by use of saliva and urine for clinical diagnostic use in general.

Isolation of subunits, alpha, beta and gamma of the complex taipoxin from the venom of Australian taipan snake (Oxyuranus s. scutellatus): characterization of beta taipoxin as a potent mitogen.

The venom of Australian taipan snake (Oxyuranus s. scutellatus) is extremely potent due to the presence of taipoxin. The intact complex molecule of taipoxin having molecular weight 45.6 kDa is composed of alpha, beta and gamma subunits. This report describes the high pressure liquid chromatography (HPLC) separation of alpha, beta (beta-1 and beta-2) and gamma subunits from taipan crude venom. The fractions containing the taipoxin subunits were further purified to obtain homogeneous proteins. The toxicity in mice showed the alpha subunit as most toxic, the gamma subunit as moderately toxic and the beta-1 and beta-2 subunits were nontoxic. The proteins beta-1 and beta-2 were found to be mitogenic having neurotrophic activity on PC12 cells in culture similar to nerve growth factor. Immunologically, alpha, beta-1, beta-2 and gamma subunits were found to be different, showing cross reactivity, and beta-1 and beta-2 were found to be identical for biological properties and molecular weight. Further characterization of unexpected mitogenic activity of beta subunits is underway.

Antigenic cross reactivity among the venoms and toxins from unrelated diverse sources.

Numerous investigators have studied and reported the antigenic reactivity of venoms from the species of snakes belonging to a genus or a family. However, there is very little published data on the inter-family antigenic cross reactivity among the venoms of snakes and absolutely no data on venoms from other sources such as honey bee, scorpion and toad. This report describes the antigenic and immunological cross reactivity among the venoms of snakes from major families: Crotalidae, Elapidae, Viperidae, Hydrophiidae and venoms from honey bee, scorpion and toad. The homologous polyclonal antisera versus snake venoms showed high reactivity to the respective venoms and varying degree to other venoms revealing the inter-family antigenic cross-reactivity. Surprisingly, venoms from bee, scorpion and toad showed antigenic cross reactivity to snake venoms. Antisera against snake venoms reacted immunologically to venoms from bee and scorpion but toad venom reacted only to anti C. atrox venom. The immunological cross reactivity among singular toxins, cobratoxin, ricin A, botulinum A and cholera was studied by using respective polyclonal antibodies. Immunological high cross reactivity was observed between bee venom and anti ricin, similarly between anti botulinum and cobratoxin. Bee venom reacted immunologically to all anti-toxins.

Detection of nerve growth factor (NGF) in venoms from diverse source: isolation and characterization of NGF from the venom of honey bee (Apis melifera).

Pearce (1973) reported the absence of NGF in the venoms of bees, scorpions, spiders, and toads. Contrary to the negative findings in the past, results of this research prove the presence of NGF in bee and scorpion venoms. Venoms from various species of snake, bee, scorpion, and toad were screened by two methods: immunological test ELISA using antibodies versus mouse NGF and venom NGF and the biological test of neurite outgrowth, the characteristic of NGF on PC cells. The presence of NGF was detected in snake, bee, and scorpion venoms, but not in toad venom by these tests. NGF was isolated from bee venom by HPLC fractionation using ion exchange chromatography. The molecular weight of bee NGF was found to be 14.0 kDa resolving into a single band by PAGE. The biological activity of bee NGF on PC12 cells was found to be 1/10 of the venom NGF.

Biological and immunological properties of nerve growth factor from snake venoms.

Homogeneous preparation of nerve growth factor (NGF) was isolated in purity by two steps HPLC fractionation from venoms of snakes belonging to the major families: Crotalidae, Elapidae, and Viperidae. Biological activity of NGF was tested on PC12 cells for neurite outgrowth and molecular weights were determined by PAGE. Antisera raised against NGFs in Balb/C mice. Immunological cross reactivity for antisera was assayed by ELISA and immunoprecipitin tests. HPLC profiles for the venoms from the species belonging to the same family were identical. The biological and immunological properties of NGFs from different species of snake belonging to the same family were also found to be identical. However, NGFs of venoms from different families of snakes showed differences in properties. Neurite outgrowth on PC12 cells due to NGF from the family Elapidae, especially the cobra species, was greater than NGF from the venoms of Crotalidae and Viperidae, with the exception of N. n. nivea which showed poor activity and C. polystictus of Crotalidae family having very good activity.

Defective parvoviruses acquired via the transplacental route protect mice against lethal adenovirus infection.

Adeno-associated virus type 1 (AAV-1) interfered with the replication of its murine adenovirus (MAV) helper in primary mouse kidney cells and in 1-day-old ICR mice. Mice carrying AAV-1 acquired via the transplacental route were protected against lethal infection with MAV. The replication of AAV-1 in these mice could be triggered by multiple challenges with MAV, and antibodies to AAV-1 were subsequently detected.

Characterization of heavy particles of adeno-associated virus type 1.

The temperature-sensitive mutant ts4 of adenovirus type 2 (Ad-2) is capable of complementing adeno-associated virus type 1 (AAV-1) in HEp2, KB and HEK cells at 34 degrees C and 39 degrees C when used as a helper virus. Heavy non-infectious AAV-1 particles can be generated by using the mutant ts4 in HEp2 cells. When AAV-1 is grown in serial passages in HEp2 cells, both the wild-type Ad-2 and the mutant ts4 give rise to heavy, less infectious AAV-1 particles. The heavy AAV-1 particles generated by Ad-2 in advanced serial passages retain the property of having CF and IF antigens, but the AAV-1 generated by the mutant in advanced serial passages lose this property. There is no appreciable difference in the particle counts made by electron microscopy of AAV-1 preparations generated either by Ad-2 or the mutant ts4. Analysis by polyacrylamide gel electrophoresis of purified heavy AAV generated by ts4 indicates that in late passage an additional polypeptide of higher mol. wt. than the three structural polypeptides is detected.

Properties of adeno-associated virus (type 1) replicated in rodent cells by murine adenovirus.

We report for the first time the replication of infectious adeno-associated virus type 1 (AAV-1) in rodent cells [primary mouse kidney (PMK) and mouse L929 cells] using murine adenovirus (MAV) as a helper virus and also the production of AAV-I virus antigen by herpes simplex virus type I (HSV-I) with its temperature-sensitive mutant ts 200 in mouse neuroblastoma (NB) cells. The infectious AAV virions produced by MAV on L cells had a buoyant density of 1.41 2ml in caesium chloride gradients.

Transplacental infection with adeno-associated virus type 1 in mice.

Adeno-associated type 1 parvovirus (AAV) was detected in the kidneys and lungs of fetuses and newborns, when pregnant mice were injected subcutaneously with AAV type 1 and murine adenovirus as a helper virus. These findings clearly indicate that transplacental infection with AAV in rodents has been achieved.