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Diagnosis of cattle fasciolosis by the detection of a circulating antigen using a monoclonal antibody
Abstract and Figures
A monoclonal antibody (MoAb) 1C12 that reacts with a 66 kDa surface tegumental (ST) antigen of adult worms of Fasciola gigantica was used to detect circulating antigen in sera of experimentally and naturally infected cattle. A combination of rabbit anti ST-antigens and MoAb 1C12 were used to capture and detect the circulating antigen in sandwich ELISA. The dilutions of 1:1,000 of rabbit anti ST-antigens and 1:100 for MoAb 1C12 were used to reduce cross-reactivity with other trematodes' antigens. The circulating antigen of F. gigantica was demonstrated in sera of all experimentally infected animals as early as the first week after the infection, and it remained detectable until the experiment was terminated at week 32 after the infection. Of the 97 serum samples from naturally infected cattle, the sensitivity of 86.6% was observed when the cut-off point was calculated from 32 serum specimens from uninfected control calves. The sensitivity increased to 100% when the commercial fetal calf and trematode-free baby calves sera were used for calculation of the control cut-off point. Based on these results, the combination of rabbit anti ST-antigens and MoAb 1C12 sandwich ELISA appeared to be sensitive, specific, and applicable in the immunodiagnosis of fasciolosis in cattle for epidemiological study and monitoring of chemotherapeutic efficacy.
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... Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. Only a few detections of circulating antigens in animals with fasciolosis have been reported with variable efficiencies due largely to their availabilities in the circulation [8][9][10][11][12][13]. ...
... It was found that these circulating antigens could be detected as early as day 1 to day 35 after infection [13]. In addition, Viyanant et al., [10] reported that a MoAbbased sandwich ELISA was used to detect circulating 66 kDa TA in the serum samples of cattle experimentally and naturally infected with F. gigantica. However, this MoAb showed crossreaction with antigens from other trematode parasites and the MoAb was unstable. ...
... For the time on antigen-detecion assays, Langley and Hillyer [8] reported that circulating antigen was detected in the sera of mice infected with F. hepatica at the 1st week of infection using rabbit antiserum. Likewise, the circulating 66 kDa antigen of F. gigantica was detected in the sera of experimentally infected cattle at the first week post infection [10]. Fagbemi et al. [9] could detect circulating 88 kDa antigen in the sera of cattle experimentally infected with F. gigantica during the 2nd and 3rd weeks after infection. ...
Background:
Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden.
Methods:
In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities.
Results:
The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test).
Conclusions:
These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.
... Hence, antigen detection is considered to be a more reliable method for identifying animals with pre-patent or occult infection, which could not be detected by the usual parasitological test. Moreover, the antigen detection can give a more accurate current rather than past infection (Langley and Hillyer, 1989;Fagbemi et al., 1995;Viyanant et al., 1997;Velusamy et al., 2004;Anuracpreeda et al., 2009a,b). ...
... In our earlier study, we have also produced MoAb against 28.5 kDa tegumental antigen (28.5 kDa TA) of adult F. gigantica and developed a sandwich ELISA for diagnosis of fasciolosis by F. gigantica in experimentally infected mice (Anuracpreeda et al., 2009b). Similarly, Viyanant et al. (1997) developed a MoAb-based sandwich ELISA for detection of circulating 66 kDa TA in the sera of cattle experimentally and naturally infected with F. gigantica. However, this MoAb cross-reacted with other parasite antigens and the MoAb clone was not stable. ...
... This early detection of CatB3 compared more favorably with other antigen-detection assays including that of Langley and Hillyer (1989) who used rabbit antiserum to detect circulating F. hepatica antigen in experimentally infected mouse sera at the 1st week post infection. As well, Viyanant et al. (1997) who could detect circulating 66 kDa TA in the sera of cattle experimentally infected with F. gigantica at the 1st week post infection. The later detections were also reported by Fagbemi et al. (1995) who could detect circulating 88 kDa F. gigantica antigen in sera of experimentally infected cattle at the 2nd and 3rd weeks after infection using a sandwich ELISA, and Velusamy et al. (2004) who could detect the circulating 54 kDa F. gigantica antigen in experimentally infected cattle sera at the 2nd week after infection. ...
A reliable monoclonal antibody (MoAb)-based sandwich enzyme-linked immunosorbent assay (sandwich ELISA) was developed for the detection of circulating cathepsin B3 protease (CatB3) in the sera from mice experimentally infected with Fasciola gigantica and cattle naturally infected with the same parasite. The MoAb 2F9 and biotinylated rabbit polyclonal anti-recombinant CatB3 antibody were selected due to their high reactivities and specificities to F. gigantica CatB3 antigen based on indirect ELISA and immunoblotting. The lower detection limit of the sandwich ELISA assay was 10, 100 and 400pg/ml, when applied for the detection of rCatB3 antigen and CatB3 in whole body (WB) of newly excysted juveniles (NEJ) and metacercariae (Met) of F. gigantica, respectively. This sandwich ELISA assay could detect F. gigantica infection from day 1 to 35 post infection and revealed that circulating level of CatB3 peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using serum samples from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as normal mice and hamsters. In addition, sera from cattle infected with Paramphistomum cervi, Strongylid, Trichuris sp. and Strongyloides sp., as well as sera from normal cattle were also assessed. In experimental mice, the diagnostic sensitivity, specificity, positive predictive value, negative predictive value, false positive rate, false negative rate and accuracy of ELISA were 95%, 100%, 100%, 97.9%, 0%, 5.3% and 98.5%, while in natural cattle they were 96.7%, 100%, 100%, 98.5%, 0%, 3.4% and 98.9%, respectively. Hence, this assay method showed high efficient and precision for early diagnosis of fasciolosis by F. gigantica.
... Sandwich ELISA based on specific mAb against F. gigantica circulating surface tegument antigen detected the antigen as early as 2 to 3 weeks post-infection. Although its sensitivity was determined to be 100%, crossreactions were observed with other trematode species (33). An in-house ELISA that used mAb against cathepsin B3 protease in the sera of naturally infected cattle yielded 96.7% sensitivity and 100% specificity (39). ...
... Sandwich ELISAs are frequently based on monoclonal antibodies (mAbs) to minimise errors caused by batch-to-batch variations common in polyclonal antibody production. The use of mAbs in this assay improves the sensitivity and specificity of immunodiagnosis (21,33). A commercialised capture sandwich ELISA, Bio K 201 kit (Bio X Diagnostics, Belgium) employing MM3 mAb for the detection of Fasciola spp. ...
Fascioliasis is an important zoonotic disease prevalent in domestic animals and it leads to socioeconomic impact in rural farming communities of the developing world. The gold standard diagnosis of ruminant fascioliasis involves coprological detection of Fasciola spp. eggs or recovery of flukes in infected livers. Coprological analysis is unreliable in the patent period of chronic infection, and even then, its sensitivity is relatively low. Robust diagnostic tools that can promptly and accurately detect an active infection are crucial to avoid complications and further losses in ruminant livestock productivity, as well as to preserve the livelihood of communities at risk. Immunodiagnosis determined by antibody and antigen detection in the sera and faeces of infected ruminants provides a valuable alternative to the parasitological diagnostic approach. This review discusses current developments in immunological techniques by enzyme-linked immunosorbent assay (ELISA) in the detection of ruminant fascioliasis and summarises the performance of various ELISAs in studies conducted to date. Indirect ELISAs demonstrated effective immunodiagnostic performance with high sensitivities and specificities. Cathepsin L ELISA is the most favourable antigen in serodiagnosis, among other recombinant and native proteins evaluated. Sandwich ELISA provides excellent sensitivity and specificity, which correlates well with the fluke burden. Utilising monoclonal antibodies in sandwich ELISA reduces the detection time and performance variations that commonly occur in polyclonal antibody ELISA.
... The SDS-PAGE analysis of Fg-E/S antigens in the present study showed that many bands have appeared, and the most prominent of which were of 15, 17, 28, 40, 60 and 84 kDa. Some bands (40, 60, and 84) were shown to be derived from the worm tegument (Viyanant et al., 1997), and they are the major recognition targets in ESP-based immunodiagnosis, while the 15,17 and 28 kDa species appeared to be released from the cells lining the gut (Anuracpreeda et al., 2006;Arjmand Yamchi et al., 2016). (Awad et al., 2009) showed that the Fg-ES antigen revealed seven protein bands with molecular weights of 15, 28, 31.6, ...
... Fegbemi et al. (1995) successfully used rabbit antibodies against the 88 kDa antigen of adult F. gigantica for the detection of circulating antigens in experimentally-infected cattle sera. Viyanant et al. (1997) studied a monoclonal antibody specific to a 66 kDa antigen of F. gigantica for the detection of circulating antigens in experimentally-and naturally-infected cattle: they reported that circulating antigens could be detected as early as the second and third weeks after infection; these antigens were associated with the crude surface tegument of the parasite. A 28 kDa protein present in E/S products of adult parasites was also identified by Ruiz Navarrete et al. (1993). ...
... Several assays utilising purified polyclonal or monoclonal antibodies that bind to Fasciola antigens have been reported and also show high sensitivity and specificity [314][315][316][317][318][319][320]. Assays have detected circulating Fasciola antigens from human [320][321][322], sheep [314,323] and cattle sera [316][317][324][325], as well as coproantigens [314, 316-318, 320-323, 326-331]. ...
The trematodes Fasciola hepatica and F.gigantica can infect a broad range of hosts, and cause the disease Fasciolosis. Fasciolosis is a disease of economic importance, with livestock infection having a considerable impact on the agriculture industry. Human infection is now considered of public health importance and is hyperendemic in some regions. The incidence of animal and human infections is increasing in parts of the world. This review will provide an overview of our current understanding of Fasciola, from distribution, diversity and disease development, to current control measures and progress towards an effective vaccine. Fasciola excrete and secrete a broad range of molecules while residing in the host. The repertoire of molecules changes as the parasite matures, reflecting the different needs of the fluke during parasite development. These molecules, which are involved in important parasite functions, are often found in the parasites excretory-secretory material, and function at the host-parasite interface. Common among these molecules are antioxidents as well as a variety of proteases, including cathepsin proteases. The roles of these key molecules during Fasciola infection, including their ability to modulate host immune responses will be a focus of the review. The findings from recent transcriptome and proteome analysis will also be a discussed, as will the potential for RNA interference in characterisation of this parasite.
... However, eventhough calf sera were used as control, our ELISA result showed cross-reaction with all schistosome sera and many sera of other parasites. Viyanant et al [13] reported that the sensitivity, when serum samples from uninfected calves was used as control cut-off value was 86.6%, but when the cut-off value was calculated from fetal calf sera and the trematode-free baby calf sera, the sensitivity improved to 100%. However, field cattle sera were not used in their study. ...
... During the last 2 decades, a major focus of research has been directed toward the identification of Fasciola antigens during human infection as a step toward the development of an efficient diagnostic assay (34). Fasciola antigens are mostly released from the gut and the tegument into the blood circulation and excreted in stools of infected animals and humans (35)(36)(37)(38)(39). Several Fasciola antigens have been detected as circulating antigens in serum or as coproantigens in feces and were successfully used in immunodiagnosis of human fascioliasis (8,11,16,34,40). ...
Currently, the laboratory diagnosis of human fascioliasis is based on the parasitological examination of parasite eggs in
stool specimens and serological detection of specific antibodies in serum samples, which are often unreliable diagnostic approaches.
Ideally, a sensitive and specific diagnostic test for Fasciola infection should be based on the detection of circulating Fasciola antigen, which implies active infection. Here, a 27-kDa-molecular-mass antigen was identified in a Fasciola gigantica adult worm antigen preparation, excretory-secretory products, and sera from F. gigantica-infected individuals, and it was not detected in antigenic extracts of other parasites and sera from noninfected individuals.
The target antigen was isolated and partially characterized as a protein. Immunoperoxidase staining located the target epitope
within teguments and guts of F. gigantica adult worms. The performance characteristics of a newly developed enzyme-linked immunosorbent assay (ELISA) based on F. gigantica circulating antigen detection in serum (FgCA-27 ELISA) were investigated using sera of 120 parasitologically diagnosed F. gigantica-infected individuals and 80 noninfected individuals. The area under the receiving operating characteristic (ROC) curve (AUC)
for ELISA was significantly high (AUC = 0.961, P < 0.0001) for discriminating Fasciola-infected and noninfected individuals. The developed assay showed high degrees of sensitivity, specificity, and efficiency
(>93%), and a significant correlation (r = 0.715, P < 0.0001) between antigen level and parasite egg count was shown. In conclusion, a 27-kDa Fasciola antigen was identified in sera of F. gigantica-infected individuals. A highly sensitive and specific Fasciola antigen detection assay, FgCA-27 ELISA, was developed for laboratory diagnosis of human fascioliasis.
... In F. gigantica, Guobadia and Fagbemi (1995) found that the 17, 21, 57 and 69 kDa protein bands were specific for F. gigantica infection in sheep. In addition, the immunoblotting of ES antigens with naturally infected cattle serum exhibited two groups of molecules (Sobhon et al., 1996): the first group consisting of molecules at 66, 64, 58 and 54 kDa believed to be the tegumental antigens, which were more species-specific than the second group which included molecules at 27 and 26 kDa that may be released from the caecum (Viyanant et al., 1997a,b). Both groups of antigens were released into the ES fraction. ...
The immunogenic components of adult Paramphistomum cervi excretion-secretion (ES) fraction were revealed by SDS-PAGE and immunoblotting technique using sera from cattle naturally infected with P. cervi, F. gigantica, strongylids, Trichuris sp., and Strongyloides sp. By SDS-PAGE, it was found that the ES fraction comprised 13 distinct protein bands. Immunoblotting analysis of these proteins exhibited 9 prominent antigenic bands which were recognized by paramphistomosis antisera. These antigenic proteins had molecular weights ranging from 10-170 kDa. One antigenic protein band of 40 kDa was found to give a consistent reaction with sera from all infected cattle. Its diagnostic sensitivity, specificity and accuracy using this test were 100%, 98.9% and 99.3%, respectively. The positive and negative predictive values were 98% and 100%, respectively. The 40 kDa antigen was partially purified by gel filtration and ion-exchange chromatography. The antigenicity of 40 kDa protein for diagnosis of P. cervi infection was confirmed by immunoblotting and indirect ELISA (at 1:78,125 dilution) using a pool of sera and individual serum samples from infected cattle. The present findings suggest that the 40 kDa protein may be used as a diagnostic antigen for paramphistomosis.
Up to now, parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Hence, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. In the present study, a monoclonal antibody (MoAb) against recombinant
Fasciola gigantica
fatty acid binding protein (rFgFABP) has been produced. As well, a reliable sandwich enzyme-linked immunosorbent assay (sandwich ELISA) has been developed for the detection of circulating FABP in the sera of mice experimentally and cattle naturally infected with
F. gigantica
. MoAb 3A3 and biotinylated rabbit anti-recombinant FABP antibody were selected due to their high reactivities and specificities. The lower detection limit of sandwich ELISA was 5 pg mL
−1
, and no cross-reaction with other parasite antigens was observed. This assay could detect
F. gigantica
infection from day 1 post infection. In experimental mice, the sensitivity, specificity and accuracy of this assay were 93·3, 100 and 98·2%, while in natural cattle they were 96·7, 100 and 99·1%. Hence, this sandwich ELISA method showed high efficiencies and precisions for diagnosis of fasciolosis by
F. gigantica
.
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
In the analysis of excretory-secretory (ES) antigens from infective third-stage larvae (L3) of Angiostrongylus cantonensis, one major component of mol.wt 91,000 was not precipitated by pooled sera of patients with eosinophilic meningoencephalitis. Monoclonal antibodies (Mc Ab) secreted from two hybridoma cell lines, established against somatic antigens of L3, recognized this molecule but with different epitope specificities indicated by an additivity index (A.I.) of 83%. The 2 Mc Ab (TD2 and 3A5) belonged to IgG2a and IgM classes, respectively. Combinations of TD2 and 3A5 were used in a sensitive enzyme-linked fluorescent assay (ELFA) for the immunodiagnosis of human angiostrongyliasis. The double-antibody sandwich ELFA method was applied firstly to sera from experimentally infected rats using either TD2 or 3A5 to coat the assay plates. Two fluorescence unit (F.U.) peaks appeared in sera from infected rats collected 18 and 44 days after infection. Specimens from 35 patients were tested, all cerebrospinal fluids (CSF) and most sera (88%) showed positive reactions and the average F.U. of CSF was greater than that of serum.
A double antibody enzyme-linked immunosorbent assay (sandwich ELISA) was used for the detection of a circulating antigen from human schistosomiasis japonica infections. This assay involves the use of polyclonal rabbit Schistosoma japonicum soluble egg antigen (SEA) antiserum to bind circulating antigen and a monoclonal antibody (MabH4) to identify and quantify this antigen. Sera from 108 S. japonicum-infected patients (acute and chronic) were tested. Sera from 93 of 95 patients with chronic infection were positive for this antigen; sera from 12 of 13 patients with acute infections were also positive. Antigen was not detectable in control human sera. Sera from 35 chronic schistosomiasis patients were collected 6-12 months after praziquantel treatment. Circulating antigen was not detectable in the sera of 33 of these patients and was dramatically reduced in 2. This ELISA system may prove valuable in differentiating past and current infections.
A 2-site enzyme immunoassay was developed for the detection of Fasciola hepatica antigen in the serum of fascioliasis infected mice. The assay utilizes high titer rabbit immunoglobulins to parasite excretory/secretory antigens (FhES) as capture antibody, and also as detection antibody when linked to horseradish peroxidase (HRP) or to biotin for reaction with avidin-peroxidase. The assays were compared with a conventional (antibody detection) ELISA to determine diagnostic utility. Using mean rates of detection of fascioliasis, the HRP-based antigen capture assay diagnosed the infection at 1 week postinfection and showed that circulating antigen levels are maximal 3 weeks after infection. The earliest mean diagnosis for the antibody detection and the biotin-based antigen capture ELISAs were 2 and 3 weeks postinfection, respectively. The addition of known quantities of FhES antigens to normal mouse serum gave estimates of lower limits of detectability for the HRP- and biotin-based assays of 25 ng and 0.25 ng, respectively. Routine use of the biotin-avidin system in the antigen capture test resulted in high background activity making this method insensitive.
We have recently developed a sensitive and specific immunodiagnostic test for canine Dirofilaria immitis infection based on detection of soluble parasite antigens in dog sera by monoclonal antibody-based enzyme immunoassay. In addition to their importance as markers of infection, these antigens may contribute to the pathogenesis of heartworm disease in dogs. In the present study, a variety of methods were used to identify and characterize circulating D. immitis antigens. Two antigens were identified in infected dog sera that formed lines of identity in rocket-line immunoelectrophoresis with soluble antigens extracted from adult D. immitis. Circulating D. immitis antigens were also demonstrated in infected dog sera by immunoblot analysis with polyclonal and monoclonal antibodies. These antigens had apparent molecular weights that ranged from 50 to 250 kDa. Most of the circulating D. immitis antigens contained the epitope defined by monoclonal antibody 1418BF2.1 which is used in our enzyme immunoassay for circulating D. immitis antigen. Studies of parasite antigens released during in vitro culture indicated that the circulating D. immitis antigens in dog sera that are detected by our enzyme immunoassay are primarily derived from adult female worms.
Antibodies to O. viverrini in the sera of people from endemic and non-endemic areas were investigated using indirect ELISA technique. For the patients from the endemic area, 92.8% who passed eggs in the stool were found to be positive for O. viverrini antibody. In addition, 46.5% of the people who did not pass eggs in the stool were also found to have low titer of O. viverrini antibody. On the other hand only 2.4% of the people from the non-endemic area with other intestinal parasite infections were found to have O. viverrini antibody in their sera. It was concluded that positive reaction of O. viverrini antibody is not cause by cross-reaction with other parasites but low liter of antibody is probably due to low-level or past infection. There is a positive correlation between the titer of O. viverrini antibody and intensity of infection as indicated by number of eggs excreted per milligram of feces. Patients with a few O. viverrini eggs in feces, but biopsy-proved-cholangiocarcinoma had very high titer of antibody.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Antibodies against a specific 88-kDa antigen of Fasciola gigantica were used for the detection of circulating antigen in the sera of cattle with experimental and natural infections of F. gigantica by a double antibody ELISA. Circulating antigen was detectable as early as the second and third weeks after infection and positive absorbance values were obtained for the entire duration of infection. Absorbance values decreased below the cutoff point 3 weeks after chemotherapy with oxyclozanide. This immunoassay also greatly enhanced the specificity of immunodiagnosis of fasciolosis in naturally infected cattle. The test system has excellent potential for the accurate diagnosis of ruminant fasciolosis.