Preparation And Identification Of Egg Yolk Immunoglobulin Against Cysticercus And The Study About Diagnosis Of Cysticercosis

Objective: Cysticercosis is an important zoonotic diseases caused by The larvae of Taenia solium ,is a focus on prevention and control of parasitic diseases. It is not only seriously endangering the health of the masses, but also causing great loss of livestock production. Therefore, strengthening the diagnosis of swine cysticercosis is important for its prevention and treatment. In this study, we clone the coding gene of the stage-specific antigen cC1 from Cysticercus cellulosae and express high levels of soluble cC1 in E.coli. 25-weeks old hens was immunized by subcutaneous injection and intramuscular injection with purified Cysticercus cellulosae cC1 protein. Then the anti-cysticercus specific yolk immunoglobulin (IgY) was prepared and identified from by cC1. We monitored the dynamic a antibody titer of IgY,and purified the antibody at higher titer. We explored cC1 protein and IgY in the the role of diagnosing swine cysticercosis by indirect ELISA and Dot-ELISA test .Methods: The recombinant plasmid pET28a-cC1 presented by Professor Fang Jiang of Bengbu Medical College was transformed into E. coli BL21. The recombinant plasmid pET28a-cC1 digested with BamH I and Xho I ,The results sent to Shanghai Sangon Biotech Technical Services Limited sequencing . The fusion expression of cC1 was induced by IPTG. The expression was subjected to SDS-PAGE followed by identification with Western blotting. The 25 week-old egg-laying HY-line hens were immunized with 200μg cC1 by subcutaneous injection and intramuscular injection with purified Cysticercus cellulosae cC1 protein for 4 times. Each hen was immunized with 200μg antigen each time, the second injection were performed 28 days later, Subsequent injection were performed at 10-day interval. The eggs layed before and after immunization were collected to make anti-cC1 IgY antibody(Storage Temperature is 4℃).IgY was extracted from the eggs by WD (water-dilution) method and purified by dialysis and salting out. The IgY was analyzed by SDS-PAGE and Western-blotting. Indirect ELISA was used to determine the titer of the antiserum. Large amount IgY was purified by EGGstractTM IgY Purification System while the antibody had a high titer. with indirect ELISA method , to examine the sera from 111 cases, including 30 normal human controls, 20 Cysticercosis patients, 30 schistosomiasis patients, 16 hookworm patients, and 15 clonorchiasis patients, and sensitivity, specificity and cross reactivity were tested respectively. and with Dot-ELISA method , to examine the sera from 111 cases, including 30 normal human controls, 20 Cysticercosis patients, 30 schistosomiasis patients, 16 hookworm patients, and 15 clonorchiasis patients, and sensitivity, specificity and cross reactivity were tested respectively.Result: The stage-specific antigen cC1 from Cysticercus cellulosae has been successfully cloned and the soluble protein efficiently expressed in E.coli, The concentration of cC1 was 3.5g/l and the molecular weight was 40ku . The molecular size of IgY detected by SDS-PAGE was equal to its predictive value. Western blotting results showed that the cC1 could be recognized by IgY collected from immunizsed hens while not by IgY collected before immunization. Using indirect ELISA, the facts were observed that the IgY antibody began to produce by hens on the fifth day after the initial immunization, and the antibody titer increased gradually over time while reached the peak with a titer overtoped 1:105 on the 55th day after immunization. There is a discrepancy on IgY antibody titer between different hens. The IgY was purified heavy by EGG stractTMIgY Purification System and the concentration was 4.96g/l by Coomassie brilliant blue . The primary detection of antibodies to cC1 in the patients with cysticercosis showed a promising sensitivity and specificity with indirect ELISA. We successfully established an indirect ELISA system to detect 20 cases of cysticercus antibodies in serum of patients with a sensitivity of 85%,specificity of 92.20%, false positive rate was 15%,false negative rate was 7.8%, positive predictive value was 68%, negative predictive value was 96.5%, Youden index was 0.772,the total agreement rate of was 90.1%, Pe was 67.57%; Kappa value was 0.6947, positive likelihood ratio was 9.67%, negative likelihood ratio was 0.16,It show that the diagnosis of cysticercosis with cC1 serum antibody has a satisfactory results. We use the Dot-ELISA to detect 20 cases of cysticercosis patients with circulating antigen in serum, the sensitivity was 95%, specificity was 98.9%, false positive rate of 5%, false negative rate of 1.1%, positive predictive value of 95 %, negative predictive value was 98.9%, Youden index was 0.939, P0 the total agreement rate was 98.2%, expected agreement rate Pe was 70.46%, Kappa value was 0.939, and clonorchiasis patients with serum, schistosomiasis hookworm disease patients infected serum and serum-free cross-reaction.Conclusion: We have successfully prepared the IgY antibody specific to cysticercus cellulosae with good security and stability. The yield of IgY is generous and easy to store. We successfully established an indirect ELISA system and the Dot-ELISA system, respectively, for the serum Cysticercus antibodies and circulating antigen detection.The preliminary tests showed that there is a satisfactory result, diagnosis of cysticercosis is expected to provide a new reagent .