Study On The Diagnostic Value Of Enolase As An Indication Of Current Infection Of Schistosoma Japonicum

Schistosomiasis is one kind of zoonosis which endangering human health servere, this disease distributes widely in 74 countries and areas worldwide. Currently, about 200 million people are suffering from schistosomiasis and 779 million people may be under threat of infection in world. There are 6 kinds of schistosoma which could parasitize in human body, Schistosomiasis japonicum is popular in P. R. China.Schistosoma infection has the feature of concomitant immunity, the immunity of anti schistosoma infection would be lost soon when the worms in the patient body were removed after effective treatment. People and animals in epidemic area could re-infection constantly as they contact the contaminated water of schistosoma cercariae repeatedly. Therefore, it will play an important role of establishing some effective diagnostic methods to find and eradicate the infection source of schistosomiasis for blocking the prevalence and harming human health of schistosomiasis. Currently, there are three kinds of experimental diagnostic methods of schistosomiasis, one is the pathogenic diagnosis based on the stool test, second is serological diagnosis based on antibody or antigen detection, the third is the molecular biological method based on detecting the nucleic acid. However, the effective prevention and control work in the past 60 years has made the prevalence of schistosomiasis in China presents the characteristics of low infection degree and low infection rate. The regular pathogenic test can not find the mild-infection patients easily, in addition, the compliance of susceptible population on stool test is poor, which limit the practical utility of these methods. Although, the sensitivity, specificity of the detecting nuclear acid methods, such as PCR, LAMP and so on, are high, the requirement of expensive equipment, rigorous experimental environment and skillful technician make these methods not be able to be used in the field of schistosomiasis epidemic area. By contrast, the serological methods are being used more widely, the traditional antibody detection methods include the indirect agglutination assay (IHA), circumoval precipitin test (COPT), enzyme-linked immunosorbent assay (ELISA), dipstick dye immunoassay (DDIA) and so on, all of them are used to detect the antibody IgG. Usually, IgG could persist in the host for a long time after treatment, it can only provide information of the patients who had a history of schistosoma infection, but not indicate the real situation of the worm burden. In addition, the rough soluble egg antigen used in these methods is apt to react with other non specific antibodies crossly, and lead to a false positive result. So the value of detecting antibody method for diagnosing and evaluating curable effect of current infection of schistosoma is poor.The circulating antigens are the antigenic substance excreted and secreted by worms parasitizing in vivo, which exists in the blood, saliva, urine and other body fluids of hosts. Detecting circulating antigens in serum or urine of patients has great value of diagnosing current infection and assessing worm loads in hosts. Because the circulating antigens in patients disappear fast after treatment, the result of detecting circulating antigen in patients can be use as an evidence of assessing the chemotherapeutic efficacy. Deelde, et al. considered that the circulating antigens could be divided into membrane related antigen (MAA), the gut related antigen (GAA) and the soluble egg antigen (SEA). The dynamic analysis showed that the three kinds of circulating antigen in hosts in serum of host could reach high peak at 10 weeks after infection. In early infection, the GAA is the major among them, but the MAA and SEA disappear faster than the GAA after treatment, which with a better value of evaluating the chemotherapeutic efficacy. However, the most of the circulating antigens is trace in serum, the efficacy of the diagnostic methods was determined by the content of detected circulating antigen target in serum of host and sensitivity of detection method. Currently, the detection methods of circulating antigen are less sensitive, and are hard to meet the need of the diagnosis and epidemiologic survey of low degree of infection and prevalence of schistosomiasis.The excreted and secreted antigens of Schistosoma japonicum adult worms (Sj ES A) were the major source of the circulating antigen, but the components of Sj ESA are very complicative, the content of its different component are different significantly. So, uncovering the components of Sj ESA and selecting the high abundant protein molecules as the detected targets would be an effective strategy of improving the sensitivity of circulating antigen detection methods. In this study, the components of Sj ESA were analyzed by the two-dimensional electrophoresis and mass spectrometry method, the Sj Enolase was determined as the highest abundant protein in Sj ESA. In order to establish a high sensitive immunologic diagnostic methods of defining the current infection of schistosoma and assessing the chemotherapeutic effect by detecting the circulating antigen, the purified recombinant Sj Enolase was prepared by adopting the geneticengineering technique, and the monoclonal antibody and polyclonal antibody against rSj Enolase were also produced. A sandwich ELISA of detecting Sj Enolase (named as Sj Enolase Sandwich ELISA) was established and optimized based on using the monoclonal antibody as capturing antibody, and using the polyclonal antibody as detecting antibody. The dynamics of Sj Enolase in sera of schistosoma infected rabbits were analyzed by optimized Sj Enolase Sandwich ELISA, and value of Sj Enolase Sandwich ELISA for diagnosis and assessing the chemotherapeutic effect of the current infection of schistosoma was investigated with the sera of low degree of schistosoma infection preliminarily.Part I Identification of the high abundant circulating antigens of Sj ESA, and the recombinant expression and characterization of Sj Enolase1. Identification of high abundant protein molecules in Sj ESAThe excreted and secretion protein of Schistosoma japonicum adult worm (Sj ESA) were prepared and analyzed by using two-dimensional electrophoresis and Coomassie Blue R-350 staining. Then the staining 2-D gel was scanned by digital scanner and the protein dots in gel were numbered, cutted out separatively and digested by trypsin. All samples of trypsinization were analyzed with Global Protein Server Work station (GPS), The searches were performed by using the NCBI non-redundant protein database with the following criteria:NCBI human database; trypsin digestion; Moxidation and iodoacetamide alkylation as the variable modifications; missed digestion site of 1; and the MS mass error of 0.1 Da. Identifications with a GPS confidence interval greater than 95% were accepted. The results of mass spectrometry showed that about 76 kinds of proteins in Sj ESA derived from Schistosoma japonicum, the high abundant protein included:2-phosphoglycerate dehydratase(Sj Enolase), 70 kDa hot shock protein, glyceraldehyde-3-phosphate dehydrogenase, fructose diphosphate aldolase, phosphoglyceric kinase, glutathione transferase, immunophilin, thioredoxin peroxidase,14-3-3 protein, PKA type Ⅱ regulatory subunit, etc.. Among them, the content of Sj Enolase was highest, which may be one of the high abundant circulating antigens.2. Gene cloning, protein expression, purification and characterization of Sj Enolase To study the diagnostic value of Sj Enolase as a circulating antigen further more, we expressed this protein in Escherichia coli. Briefly, the mRNA of adult worms of Schistosoma japonicum was extracted with Timesaver mRNA purification system following the manufacturer’s instructions, and the first strand cDNA was synthesized with the SuperScript Choice System. The specific primers for amplifying the the DNA fragment of open reading frame encoding the Sj Enolase protein were designed according to the published sequence (gi46201125). A BamH I site was introduced into the 5’end of the upstream primer, and a Sal I site was introduced into the 5’end of the downstream primer. The DNA fragment encoding Sj Enolase was amplified from S. japonicum adult worm cDNA by phusion high-fidelity enzyme, and cloned into a TA cloning vector to construct the recombinant plasmid pGEMT-SjEnolase. The inserted Sj Enolase DNA fragment was confirmed by restrictive analysis and DNA sequencing, and then subcloned into the expression plasmid pET28a (+) at the BamH I and Sal I restriction sites to form the recombinant plasmid pET28a-SjEnolase. This recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells, and positive transformants were selected on a Luria-Bertani (LB) media plate containing kanamycin (50μg/mL) and induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) at different temperature to express the Sj Enolase protein. The expressed products were analyzed by SDS-PAGE showed that a 50 kDa soluble recombinant Sj Enolase (rSj Enolase) was expressed in the expression product when the bacteria of stationary phase (OD600=2.4) were induced with 0.1 mM IPTG at 18℃, and the recombinant Sj Enolase protein was purified with nickel-nitrilotriacetic acid (Ni-NTA) resin following the manufacturer’s instruction.For observing the immunogenicity of rSj Enolase,5 ICR mice (6 week old, female) were immunised, the level of serum antibody against rSj Enolase was over 1:10,000, which suggested that rSj Enolase has a stronger immunogenicity. The results of Westernblotting showed that the immunized serum could recognized the natural Sj Enolase in adult worm protein, the rSj Enolase also could be recognized by the sera of schistosomiasis patients, severe infection rabbits and mice, but can not be recognized by the sera of healthy people, rabbits, mice and clonorchiasis, which indicated the rSj Enolase has a natural immunogenicity and immunoreactivity, and with a good potential of immunodiagnosis.Part 11 Preparation and characterization of antibody against rSj EnolasePreparation of the polyclonal antibody against rSj Enolase Rabbits were immunized with complete adjuvant antigen of the rSj Enolase, each rabbit was injected intradermally with 2 ng protein in neck skin, and boosted with incomplete adjuvant antigen of the rSj Enolase in he same quantity of protein for three times, with a interval of 2 weeks each. The sera of immunized rabbits were collected by Carotid artery bleeding after two weeks post final immunization. The titer of serum antibody against the rSj Enolase was higher than 1:200000, which was determined by the universal ELISA method. The polyclonal antibody IgG was purified by affinity chromatography with Protein G Sepharose resin from the immunized rabbit serum, and its affinity constant was 2.7×108 mol/1 determined by non competitive ELISA.Preparation of the monoclonal antibody against rSj Enolase Mice (BALB/c, female,8 week-old) were immunized with complete Freund antigen of rSj Enolase (50μg) intradermally in the first immunization, then boost three times with the same amount of rSj Enolase incomplete adjuvant antigen with a interval of 2 weeks each, final boost was injected the same dose of rSj Enolase in PBS into mice abdomen after one week post the fourth immunization. At the last week post the last immunization, the single spleen cells were prepared from immunized mice spleen, and were fused with SP2/0 cell at logarithmic phase in the proportion of 6:1 by 50% PEG-4000. The fusion cells were seeded in 96 wells plates with HAT selective RPMI 1640 medium. The medium was changed every week at half the amount with HAT and the Hybridoma cells in each well were monitored by microscopic examination. The wells in which the hybridoma secreted the specific antibody against Sj Enolase were selected by ELISA and the hybridoma cells were subcloned by limit dilution method for several times until the specific antiobody against Sj Enolase of all wells became positive, then those cells which could secrete the monoclonal antibody steadily in a long term were stored in liquid nitrogen.5 strains of hybridoma cells were obtained, which were named after 13G8、 13A3、11F6、4G3、and 1G9, respectively. The subclasses of each strain was IgG1、IgG1、 IgG2a、IgG1 and IgG2b, respectively and the titer of antibody against Sj Enolase in the supernatant of culture medium was 1:200000,1:120000,1:100000,1:150000,1:100000, respectively.Both titers of ascites from mice innoculated with hvbridoma 13G8.4G3 respectivelv were over 1:204800. The affinity constants of monoclonal 13G8,4G3 were also determined through non competitive ELISA, which is 8.2×107 mol/l,4.87×107 mol/l, respectively. The results of Western blotting showed that McAb 13G8 could recognize both the rSj Enolase and the natural Sj Enolase of SEA and AWA, but could not cross react with the protein of bacterium E. coliBL21 and Clonorchia sinensis, which indicated that the monoclonal antibody 13G8 was high specific.Part Ⅲ Establishment and optimization of double-antibody sandwich ELISA for detecting Sj EnolaseTo construct a double antibody sandwich ELISA for detecting the circulating antigen Sj Enolase (named as Sj Enolase sandwich ELISA), the purified monoclonal antibody or polyclonal antibody was used as the capturing antibody of circulating antigen Sj Enolase, and purified rabbit polyclonal antibody or monoclonal antibody was used as the detection antibody of circulating antigen Sj Enolase respectively, the HRP labeled goat anti rabbit IgG or HRP labeled goat anti mouse IgG was used as the second detection antibody, and substrate 3,3’,5,5’-tetramethylbenzidine (TMB) was used for color development. In accordance with the method of the checkerboard phalanx, the optimized antibody matching of the sandwich ELISA, the most favorable working concentration of the captured antibody and detection antibody were determined. On these bases, the optimized condition of the capturing antibody coating, the capturing antibody reacts with antigens, antigen reacts with detection antibody, the enzyme conjugate responding codition and the color developing condition of substrate were determined used the same method as above. Finally, the optimized double-antibody sandwich ELISA for detecting Sj Enolase was established, the reaction volume is 100μL. The specific monoclonal antibody was used as the capturing antibody, the optimized coating concentration was 6.25 μg/mL, the polyclonal antibody, its optimized working concentration was 1.25μg/mL; The favorable test condition of the sandwich ELISA is:the wells of ELISA plate were coated with the capture antibody at 4℃ overnight and blocked with PBS containing 5% skimmed milkpower at 37℃ for 1h. The plate was washed three times with PBST (containing 0.05% Tween-20), and then the circulating antigen solution containing Sj Enolase or serum samples were added into the wells. The plate was incubated at 37℃ for 2 h, and then reacted with detection antibody at 37℃ for 1 h after washing. The plate was washed three times again and reacted with HRP labeled goat anti-rabbit IgG diluted by 1:10,000 at 37 ℃ for 1 h. The plate was washed with PBST three times again; 50μL of the substrate TMB solution was added into each well of the plate. The plate was kept at room temperature for 5 min to develop color and stopped with 50μL of 2 M sulfuric acid. The value of OD450nm of each well was measured at 450 nm by an ELISA plate reader. The 2.1 times of OD450 value of the blank control well was used as the cut off value, which was 0.192. The detection limit of this method was700 pg/mL and the linear range covered from 0.7ng/ml to 1000 ng/mL.Part IV The dynamics of Sj Enolase in infected animal and its value of diagnosis and evaluating the chemotherapeutic effect of schistosomiasisThe specific monoclonal antibody was used a detection antibody, the dynamics of Sj Enolase in the serum of the same rabbit at the different time points before the infection, after the infection and after praziquantel treatment were observed by Dot-ELISA. The results showed no Sj Enolase in the sera of healthy rabbits before schistosoma infection, but the quantity of Sj Enolase in infected rabbits increased gradually, and kept a high level until the 18th week post infection in the untreated group. While in the treated group, the quantity of Sj Enolase in rabbits increased continually in the first 2 weeks after praziquantel treatment, then decreased rapidly, and reached to a negative level at the 12-16th week after praziquantel treatment. These results were similar to the one of the Sj Enolase sandwich ELISA. These results suggested that the circulating antigen Sj Enolase in the serum of rabbit was related to the Schistosoma japonicum worm presenting in vivo.The serum samples of schistosomiasis (including 45 matching sera from the same patient before treatment and at different time points after treatment), paragonimiasis, clonorchiasis and the healthy were detected in parallel by the double-antibody sandwich ELISA and SEA-ELISA which for detecting the specific antibody IgG against SEA. The cut-off threshold was determined by the area under the ROC curve, the sensitivity (SE), specificity (SP), positive predictive value (PPV), negative predictive value (NPV) and Youden Index of this method was calculated. The value of diagnosis and assessing the chemotherapeutic effect of Sj Enolase sandwich ELISA and SEA ELISA was explored respectively by observing the negative rate of Sj Enolase and antibody IgG against Sj Enolase in the serum of curable patient of schistosomiasis. Among 143 sera of schistosomiasis,120 sera were positive,92 of 96 sera of healthy were negative, the sensitivity and specificity of the Sj Enolase sandwich ELISA was 84.61 percent, and 95.83 percent, respectively, Youden Index was 0.8. The positive predictive value (PPV) and negative predictive value (NPV) was 95.31 percent and 86.17 percent, respectively.The matching sera from 45 schistosomiasis patients, which included the sera before treatment, and at the 3th month,6th month,9th month,12th month after praziquantel treatment were detected by the double-antibody sandwich ELISA. The positive rate was 89.58% before praziquantel treatment, but the negative rate in the 3th month,6th month,9th month,12th month post praziquantel treatment reached to 93.33%,97.78%,100% and 100%, respectively. The content of Sj Enolase in the sera of patients was positively correlative to the infection degree of schistosoma (r=0.768, p=0.000). These results indicated that the circulating antigen Sj Enolase disappeared along with the elimination of worms in patients through an effective treatment. It is of a great value to detect the circulating antigen Sj Enolase for diagnosis and evaluating the chemotherapeutic effect of schistosomiasis. Among 30 sera of clonorchiasis, only one serum was positive, the cross reaction rate was 3.33%. Among 20 sera of paragonimiasis, only one serum was positive, the cross reaction rate was 5%. Nevertheless, the matching sera from 45 schistosomiasis patients were detected by SEA ELISA, the positive rate was 95.56% before praziquantel treatment, and the negative rate in the 3th month,6th month,9th month, and 12th month post praziquantel treatment was 28.89 percent,28.89 percent,37.78 percent and 42.22 percent, respectively. The cross reaction rate of SEA ELISA with clonorchiasis and paragonimiasis was 16.67 percent and 65 percent, respectively.ConclusionIn this study, the Sj Enolase was confirmed as the most abundant protein in ESA and may be a high abundant circulating antigen. The established Sj Enolase Sandwich ELISA with high specificity and sensitivity could be used to detect the patients with low degree of schistosoma infection, the test results of this method could reflect the infection degree of Schistosoma japonicum and could be used to evaluating the chemotherapeutic effect, which would be expected to make up for the lack of the effective diagnosis method in schistosomiasis prevention and control work of low degree prevalence.