adult worm of echinococcus granulosus lives in small intestine of carnivores , and infected eggs are excreted in their feces . following ingestion of eggs by the intermediate host ( herbivores and humans ) , larval stage of the parasite , hydatid cyst , develops in internal viscera . clinical signs and symptoms of the disease are not specific and depend on the location of the cyst . imaging techniques are able to identify the cyst in the body , but they are not able to differentiate the cyst from benign or malignant tumors . moreover , the infection may be associated with other infections such as fungal infection . in these cases , also , differential diagnosis is important . different immunological methods including casoni test , complement fixation , indirect hemagglutination , latex agglutination , enzyme - linked immunosorbent assay ( elisa ) , and immunofluorescent methods have been used for this differential and specific diagnosis . in previously published works , this variability may be due to the type of antigen and geographic region of the parasite . western immunoblotting which provides fractionation of the parasite antigens may provide higher sensitivity and specificity . a few works have been performed using western immunoblotting in diagnosis of human hydatid cyst . sensitivity of 72%97% and specificity of 96%100% have been reported for this test in diagnosis of hydatidosis . considering this fact that certain genotype of the parasite exists in certain area of the world , in this work , diagnosis of hydatid cyst with western immunoblotting technique using antigens from local sheep hydatid cyst has been investigated . in this cross - sectional study , e. granulosus hydatid cysts were collected from sheep from a slaughter house in isfahan , iran . hydatid fluid was aspirated from sheep hydatid cyst , and with observation of the protoscolices , the tubes containing fluids were centrifuged at 2000 g , for 2 min , and the supernatant was stored at 20c as hydatid fluid antigen . the sediment which was compact of many protoscolices washed with isotonic saline , sonicated and then kept at 20c as crude protoscolex antigen . laminated and germinal layers of the cyst were removed , homogenized , sonicated , and then kept at 20c as cyst wall antigen . polyacrylamide gel electrophoresis with sodium dodecyl sulfate and western immunoblotting were performed using bio - rad apparatus according to the methods of laemmli and towbin et al . briefly , antigens run on 15% acrylamide gel under reducing conditions and transferred to nitrocellulose papers . the papers were then probed with different sera at appropriate concentrations . following washing with the buffer , finally , appropriate substrate was added to develop reaction of sera with the bands of antigens . according to the previous investigation , sample size of thirty was estimated for this work , so spare sera of thirty patients with hydatid cyst were collected when they were subjected to surgery for removing their cysts . for control group , sera of thirty blood donors whose fecal examinations were negative for parasitic infection were collected from blood bank . sensitivity , specificity , positive predictive value , and negative predictive value of western immunoblotting in diagnosis of human hydatid cyst were estimated considering results of surgery as gold standard . hydatid cyst fluid ( hcf ) antigen on western immunoblotting was probed with thirty individual sera of patients with hydatid cyst and also thirty normal sera . of 30 sera , 29 ( 96.6% ) however , none of the normal sera reacted with that band [ figure 1 ] . when protoscolex crud antigen was probed with sera of thirty patients with hydatid cyst or thirty normal sera , 26 ( 86.6% ) of 30 hydatid cyst patients sera reacted with a band about 7 kda in western immunoblotting . again , none of the normal sera reacted with this band [ figure 2 ] . using cyst wall crud antigen , 16 ( 53.3 ) of 30 sera of patients with however , none of the normal sera reacted with this band [ figure 3 ] . western immunoblotting of hydatid cyst fluid probed with the sera of patients with hydatid cyst ( 17 ) or normal human sera ( 8 and 9 ) . m stands for molecular weight marker western immunoblotting of protoscolex probed with the sera of patients with hydatid cyst ( 17 ) or normal human sera ( 8 and 9 ) . m stands for molecular weight marker western immunoblotting of cyst wall probed with the sera of patients with hydatid cyst ( 17 ) or normal human sera ( 8 and 9 ) . m stands for molecular weight marker sensitivity , specificity , positive predictive value , and negative predictive value of western blotting for diagnosis of hydatid cyst infection were estimated considering the result of surgery as gold standard . when protoscolex antigen was used , sensitivity of 86% and specificity of 100% was achieved . with cyst wall antigen , results of western blotting test for thirty serum samples of hydatid cyst patients and thirty serum samples of healthy controls with different antigens are summarized in table 1 . in addition , sensitivity , specificity , positive predictive value , and negative predictive value of western immunoblotting for diagnosis of hydatid cyst are summarized in table 2 . kappa coefficient index for hcf , protoscolex , and cyst wall antigens was 96.6% , 86.4% , and 53.4% , respectively . results of western immunoblotting of hydatid cyst antigens ( hydatid cyst fluid , protoscolex , and cyst wall ) probed with thirty sera of patients with hydatid cyst and thirty sera of healthy controls sensitivity , specificity , positive predictive value , and negative predictive value of western immunoblotting in diagnosis of human hydatidosis in this investigation , 96.6% of sera of patients with hydatid cyst reacted with 20 kda band of cyst fluid and 53.3% of sera also reacted with a 7 kda band of cyst wall . moreover , 86.6% sera of patients with hydatid cyst reacted with a 7 kda of protoscolex crud antigen . . however , those techniques are not able to differentiate hydatid cyst from other disorders such as benign or malignant tumors . immunological methods would be very beneficial for this differential diagnosis . hence , different immunological techniques such as elisa have been developed for this differential diagnosis . in elisa test , hcf antigens including antigen 5 ( ag5 ) and agb and also crude liquid antigen have been used for immunological diagnosis of hydatid cyst . using crude antigen , sensitivity of 64%100% and specificity of 41%100% was achieved . with agb , sensitivity of 30%92% and specificity of 77%97% was detected . with arc5 as antigen , sensitivity of 70%100% and specificity of 70%100% was reported . in western immunoblotting , different antigens including hcf , agb , and e. granulosus 19 kda have been used for diagnosis of hydatid cyst . they reported a sensitivity of 72% and specificity of 100% for this test . in another work , jiang et al . reported a sensitivity of 91% and specificity of 92% for this test . finally , doiz et al . used sheep hydatid fluid antigen for diagnosis of human hydatid cyst . they showed that western blotting was able to detect 100% of cases of hydatidosis . in our investigation , we achieved sensitivity of 96% and specificity of 100% for hcf , 86% and 100% for protoscolex , and 53% and 100% for cyst wall antigen . result of our study is in agreement with results of other works for western blotting technique . the main problem with the results of the different tests in diagnosis of hydatidosis is existence of a wide range of variation for reported sensitivity and specificity . this variation may be related to the sample size of the study or batch of the antigen which has been used in the given tests . in our work , when hcf used as antigen , sera of patients with hydatid cyst reacted with different bands . regard , doiz et al . reported that sera of patients with hydatid cyst reacted with bands with molecular weight of 12 , 14 , 16 , 20 , 24 , 34 , and 39 kda of hcf . in our study , both sera of patients with hydatid cyst and normal sera reacted with different bands of cyst wall . in this regard , it has been shown that bands with molecular weight of about 55 , 25 , and 29 kda of laminated layer reacted with anti - sheep igg . results of this investigation revealed that 96.6% of sera of patients with hydatid cyst reacted with a 20 kda band of hcf . considering results of surgery as gold standard , sensitivity of 96% and specificity of 100% was achieved for western blotting . hence , the 20 kda band of hcf might be a good candidate for diagnosis of human hydatid cyst and more work in this regard is recommended . mh : performance of lab worksmn : providing human samples and contribution in designing the research projectbs : providing human samples and contribution in writing the manuscriptzg : contribution in designing the research project and writing the manuscriptsms : contribution in lab works and writing the manuscripthy : contribution in lab works and writing the manuscripthyd : designing research project , supervision of lab work , and writing the manuscript . mh : performance of lab works mn : providing human samples and contribution in designing the research project bs : providing human samples and contribution in writing the manuscript zg : contribution in designing the research project and writing the manuscript sms : contribution in lab works and writing the manuscript hy : contribution in lab works and writing the manuscript hyd : designing research project , supervision of lab work , and writing the manuscript .
background : echinococcosis is a parasitic disease with worldwide distribution which is caused by the tapeworms echinococcus granulosus . 
 diagnosis of the disease relies on imaging techniques , but the techniques are not able to differentiate the cyst from benign or malignant tumors ; hence , appropriate serologic methods are required for the differential diagnosis of the infection.materials and methods : in this investigation , different sheep hydatid cyst antigens probed with thirty sera of patients with hydatid cyst and also thirty human normal sera using western immunoblotting technique . 
 considering results of surgery as gold standard , sensitivity and specificity of western blotting was estimated.results:sera of 29 , 26 , and 16 patients with hydatid cyst reacted with specific bands of hydatid cyst fluid ( hcf ) , protoscolex crude antigen , and cyst wall crude antigen , respectively . 
 however , none of the normal human sera reacted with those specific bands.conclusion:a 20 kda band of sheep hcf is an appropriate antigen for serodiagnosis of hydatid cyst infection .