| Trichinellosis is a serious parasitic zoonosis mainly caused by eating raw or undercooked pork and the meat of other animals containing Trichinella spiralis larvae. It is estimated that 11 million people in the world are infected by Trichinella. More than 10 000 cases of human trichinellosis were reported by the International Commission on Trichinellosis (ICT) from 1995 to 1997. Since human trichinellosis was found in China in 1964, 548 outbreaks of this disease has been occurred 12 Provinces/Autonomous Regions/Municipals (P/A/M) of China. Henan province is one of the high endemic areas of trichinellosis. Trichinellosis not only harms to human bodies but also causes huge economic loss. However, there are not satisfactory diagnostic techniques for this disease at present. The confirmed diagnosis relies on the muscle biopsy, but the larvae can not be detected in patients with light infection or in the earlier stage of infection, and the muscle biopsy is not easily accepted by patients. Hence, in order to resolve this problem, a lot of studies on immunodiagnosis of trichinellosis have been carried out at home and abroad. But, most of diagnostic antigens used in these studies were crude antigens of the encysted (muscle stage) larvae of T. spiralis. Because the antigenic components are very complicated, when the antigens were used for the immunodiagnosis of trichinellosis, they often occurred cross-reactions with sera from patients with other parasitic diseases (for example, paragonimiasis, clonorchiasis, schistosomiasis japonicum, cysticercosis and so on) and result in false-positive reactions. So it is very difficult to make a specific diagnosis for trichinellosis. In order to find specific antigens for the diagnosis of trichinellosis, the components of T. spiralis muscle larval soluble antigens and its excretory-secretory (ES) antigens were firstly analyzed by SDS-PAGE technology, then these antigens were used to react with sera from patients with trichinellosis, other other parasitosis and normal rats, mice and persons by using immuno-blotting. In addition, in order to identifythe antigen causing cross reaction, adult worm soluble antigens of Paraonimus westermani and Clonorchis sinensis were analyzed too.T. spiralis muscle larva, P. westermani adult and C. sinensis adult were homogenized, ultrasonicated, stirred, frozen and melted repeatedly, centrifugated, respectively, the supernate was collected and used as the worm soluble antigens. T. spiralis larvae were cultivated in serum-free RPMI-1640 medium for 18, 21, 24, 30 hours at 37C in CO2 incubator respectively. The culture fluids were collected, centrifugated, dialysed, lyophilized, respectively and T. spiralis muscle larva ES antigen were obtained. Protein contents of each antigens were assayed. These antigens were analyzed by the SDS-PAGE. 11% acrylamide separating gel and 4% acrylamide stacking gel were used in the electrophoresis. The gel was stained and bleached sufficiently, different electrophoresis bands were revealed. According to the migrating rate of standard molecule weight protein bands and the logarithm of molecule, standard curve was drawn, and then the corresponding molecule weight was checked out. Each antigens were analyzed by Western blot.The results of SDS-PAGE and Western blot were described as follows.1. Choice of protein contents of electrophoresis sample: there were differences among the electrophoresis bands, when the protein concentration was different in sample hole. The higher the sample concentration is, the less the quantity is needed, the better the bands is revealed, when electrophoresis system of Bio-Rad company was used, the protein content was about 14ug/hole; when electrophoresis system of Chinese company used, the protein content was about 25ug/hole. The amount of sample filled 1/4 of the hole, better results were acquired.2. Analysis of T. spiralis muscle larva soluble antigen: muscle larva soluble antigen revealed the complicated band pattern consisted of 29 protein bands. The major bands with mole... |
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