| Background:Serum therapy is the primary treatment for systemic envenomation by D. acutus. Three types of D. acutus antivenom are produced and available in China: liquid antivenom from Shanghai Institute of Biological Products, lyophilized antivenom from Cheng-Du Military Area Center of Disease Prevention and Control, and lyophilized antivenom from the National Institute of Preventive Medicine, Taipei, Taiwan. Antivenom is isolated from the plasma of adult horses immunized with formaldehyde-detoxified D. acutus venom. Although formaldehyde or glutaraldehyde-treated venom can produce high-titer antiserum[1,2,3], repeated immunization of the formaldehyde- or glutaraldehyde-containing solution inevitably harms the horse. Venom detoxification methods are needed that produce antivenom with higher immunogenicity and lower toxicity. Several improved techniques for venom detoxification have been reported, including titration of native venom with iodine monochloride solution[4], encapsulation of native crotoxin in liposomes[5], irradiation of toxic proteins with gamma-rays[6,7], and selective heat denaturation[8,9]. Heat denaturation is the simplest detoxification technique and it has the ability to reduce venom toxicity without altering the immunogenicity of components with molecular weights lower than 25 kDa[8,9].Objectives:The aim of this study was to assess the effects of various temperatures on the precipitation, immunogenicity, biological toxicity, and interactions of different toxic components from D. acutus venom, and improve toxoid preparation.Methods:1. Separation of fractionsThe D. acutus venom was separated by gel filtration chromatography (Sephadex G-50), eluted by distilled water with a flow rate of 2.0 ml/min at room temperature (25oC). The target fractions were pooled and lyophilized. Then their LD50 and hemorrhage acticity were determined.2. Determination the critical temperature of venom and it fractionsThe venom and its fractions (peak 1 and peak 2) were heated to various temperatures (45oC–70oC) for 30 min, after which immunoreactivity and hemorrhagic activities of the samples were determined. According to the critical temperature for immunoreactivity and hemorrhagic activities, selected an optimizing temperature, at which the toxicity of venom and its fractions was significantly reduced without significantly altering immunoreactivity.3. Biological toxicity of heated (optimizing temperature) and non-heated samplesThe venom and its fractions (peak 1 and peak 2) were heated at optimizing temperatures for 30 min, after which lethality, myotoxicity activities, hemorrhagic activities and membrane lysis activities of the samples were determined and compared with the non-heated samples.4. Synergistic toxic effect of peak 1 and peak 2Peak 1 fraction and peak 2 fraction was injected i.m. into the gastrocnemius muscle of the mouse'hind leg through two experimental protocols: In protocol A (peak 1/peak 2 group), the peak 1 fraction was injected into the right hind leg, and the peak 2 fraction was injected into the left leg; In protocol B (peak 1 + peak 2 group), the peak 1 fraction was mixed with the peak 2 fraction in equal lethal doses, and the same mixture was injected i.m. into the both hind legs. After injection, each animal's survival time was recorded immediately after respiration ceased, or their blood samples were collected 90 min after injection of fractions, and the serum CK activities were detected.5. Polyclonal antibodies to D. acutus venomThe hartley guinea pigs were immunized with attenuation fraction (peak 1 fraction and peak 2 fraction heated at optimizing temperature) by two protocols, one is two fractions injected together at the same position, the other is two fractions injected at different sites. The animal immunized with non-heated crude venom was control group. At the end, compare titers and protective abilities of the antiserum of the three groups.6. Statistical analysisResults were presented as mean±standard deviation. The significance of difference between two independent samples was evaluated by Student's t-test. In all analyses, the level of statistical significance chosen was P < 0.05. Results:1. The venom of D. acutus was separated into three fractions with a Sephadex G-50 column and named peak 1, peak 2 and peak 3. Venom exhibited at least 13 distinct bands on non-reducing SDS-PAGE, ranging from 12 to 105 kDa. Comparing the proteins of peak 1 with peak 2, the higher molecular weight bands (> 21 kDa) were located only in peak 1, while the lower molecular weight bands (< 21 kDa) were mainly located in peak 2. It was strange that the molecular weight of peak 3 was about 30 kDa, which was larger than peak 2.2. The optimizing heating set points of crude venom, peak 1 fraction, and peak 2 fraction were approximately 60oC, 55oC, and 60oC. Compared with non-heated venom and its fractions, the biological toxicity (lethal, hemorrhagic, vitelline membrane lysis, and myotoxic activities) of venom and its fractions heated to the critical temperature for 30 min was significantly decreased, while their immunoreactivity and immunogenicity was maintained.3. The synergistic toxic effect of fractions (peak 1 and peak 2) was determined by comparing the serum CK and mean survival time between the two treatment groups (peak 1/peak 2 vs peak 1 + peak 2). After injection, mean survival time of the peak 1 + peak 2 group was shorter than the peak 1/peak 2 group. Similarly, higher myotoxic activity (higher serum CK value) was observed in peak 1 + peak 2 group.4. The antibodies elicited by heated fractions and non-heated crude venom were able to recognize the native venom in ELISA, but the antiserum of crude venom had higher antibody titers than other two groups, and the peak 1/peak 2 group have a higher titer than peak 1+peak 2 group. However, the antiserum from peak 1/peak 2 group was the most effective in neutralizing the lethality induced by the crude venom at the tested dose.Conclusions:1. Separating the snake venom into fractions, and selecting heat denaturation temperature of each fraction can significantly reduce the toxicity of venom fractions without significantly altering immunoreactivity. This could improve the potency of snake antiserum.2. When immuned animal to prepare antivenomous serum, injecting different fractions into different sites to avoid the synergistic toxic effect of fractions can produce high potency and effective antiserum. 3. The yolk membrane lysis detect, which invented by ourselves, can actually detected the lysis of snake venoms or other biotoxins. |
PDF Full Text Request