scholarly journals Combined Use of Indirect ELISA and Western Blotting with Recombinant Hepatocellular Carcinoma-Associated Antigen 59 Is a Potential Immunodiagnostic Tool for the Detection of Prepatent Haemonchus contortus Infection in Goat

Animals ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 548 ◽  
Author(s):  
Muhammad Ali-ul-Husnain Naqvi ◽  
Sana Zahra Naqvi ◽  
Muhammad Ali Memon ◽  
Kalibixiati Aimulajiang ◽  
Muhammad Haseeb ◽  
...  
Keyword(s):  
Western Blot ◽  
Western Blotting ◽  
Haemonchus Contortus ◽  
Indirect Elisa ◽  
Fasciola Hepatica ◽  
False Negative ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Combined Use

Haemonchus contortus is recognized as one of the important health problems in small ruminants, leading to reduced production and economic loss for farmers worldwide. Prepatent diagnosis of H. contortus infection is crucial to improve control strategies as this helminth may remove up to one-fifth of total erythrocytes and may cause anemia, edema, diarrhea, and ultimately death in young animals. In this study, one of the excretory and secretory products, rHc-HCA59, was purified and used as antigen to detect specific antibodies in H. contortus infected goats during prepatent stage of infection using indirect enzyme linked immunosorbent assay (ELISA) as screening test. All goats (n = 38) were housed indoor, experimentally infected with 8000 infective larvae (L3) of H. contortus, and serum samples were collected prior to infection and at 14th day of infection. Immunoblotting was performed to confirm the results of indirect ELISA, evaluate the cross reactivity against rHc-HCA59 in sera of most common co-infecting parasites and rectify the false negative samples. Furthermore, three different batches of rHc-HCA59 were produced to evaluate the repeatability of ELISA. No eggs were detected in feces of all goats collected at 7th and 14th day of infection but, H. contortus eggs were detected at 21 days post infection in the feces. Indirect ELISA performed in this study showed 87% sensitivity and 100% specificity. The western blot analysis confirmed immunoreactivity in serum samples which scored positive in indirect ELISA and recognized the samples as negative which had OD450 lower than negative cut-off value in indirect ELISA. Furthermore, all false negative sera (n = 5) that had OD450 value between positive and negative cut-off value in rHc-HCA59 based ELISA were clearly positive in western blot. Moreover, no cross-reactivity was detected in ELISA and western blotting against rHc-HCA59 in positive sera of Toxoplasma gondii, Fasciola hepatica, and Trichinella spiralis. The results of this study concluded that combined use of indirect ELISA and western blotting with rHc-HCA59 is a potential immunodiagnostic tool for the detection of H. contortus infection during prepatent period in goats.

scholarly journals Comparison between available serologic tests for detecting antibodies againstAnaplasma phagocytophilumandBorrelia burgdorferiin horses in Canada

2015 ◽  
Vol 27 (4) ◽  
pp. 540-546 ◽  
Author(s):  
Gili Schvartz ◽  
Tasha Epp ◽  
Hilary J. Burgess ◽  
Neil B. Chilton ◽  
Katharina L. Lohmann
Keyword(s):  
Point Of Care ◽  
Fluorescent Antibody ◽  
Clinical Status ◽  
False Negative ◽  
Pairwise Comparison ◽  
Cross Reactivity ◽  
Multiplex Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Serologic Tests

To investigate the agreement between available serologic tests for the detection of antibodies against Anaplasma phagocytophilum and Borrelia burgdorferi, 50 serum samples from horses of unknown clinical status and at low risk for infection were tested. In addition to a point-of-care enzyme-linked immunosorbent assay (pocELISA), the evaluated tests included 2 indirect fluorescent antibody tests (IFATs) for antibodies against A. phagocytophilum and an IFAT, an ELISA confirmed with Western blot, and the Lyme multiplex assay for antibodies against B. burgdorferi. For each pair-wise comparison between serologic tests, the difference in the proportion of seropositive results as well as kappa and the prevalence-adjusted, bias-adjusted kappa were calculated. The proportion of seropositive results differed significantly in each pairwise comparison of tests for detection of antibodies against A. phagocytophilum, and between the pocELISA and IFAT as well as between the pocELISA and Lyme multiplex assay for detection of antibodies against B. burgdorferi. Agreement based on kappa varied from poor to fair while agreement was improved when evaluating prevalence-adjusted, bias-adjusted kappa. Lack of agreement may be explained by differences in methodology between the evaluated tests, cross-reactivity or false-positive and false-negative tests. In addition to the limitations of serologic test interpretation in the absence of clinical disease, this data suggest that screening of horses for exposure to tick-borne diseases in nonendemic areas may not be warranted.

scholarly journals Optimization and Validation of Indirect ELISA Using Truncated TssB Protein for the Serodiagnosis of Glanders amongst Equines

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Harisankar Singha ◽  
Praveen Malik ◽  
Sachin K. Goyal ◽  
Sandip K. Khurana ◽  
Chiranjay Mukhopadhyay ◽  
...  
Keyword(s):  
Indirect Elisa ◽  
Expression System ◽  
Cross Reactivity ◽  
Amino Acid Sequences ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Diagnostic Efficacy ◽  
Diagnostic Potential ◽  
Prokaryotic Expression System ◽  
Control Serum

Objective. To express truncated TssB protein ofBurkholderia malleiand to evaluate its diagnostic efficacy for serological detection of glanders among equines.Materials and Methods. In an attempt to develop recombinant protein based enzyme-linked immunosorbent assay (ELISA), N-terminal 200 amino acid sequences ofB. malleiTssB protein—a type 6 secretory effector protein—were expressed in prokaryotic expression system. Diagnostic potential of recombinant TssB protein was evaluated in indirect ELISA using a panel of glanders positive (n=49), negative (n=30), and field serum samples (n=1811). Cross-reactivity of the assay was assessed with equine disease control serum and human melioidosis positive serum.Results. In comparison to CFT, diagnostic sensitivity and specificity of ELISA were 99.7% and 100%, respectively.Conclusions. The indirect ELISA method using the truncated TssB offered safer and more rapid and efficient means of serodiagnosis of glanders in equines. These data highlight the use of TssB as potential diagnostic antigen for serological diagnosis of glanders.

scholarly journals Mycoplasma agassizii Strain Variation and Distinct Host Antibody Responses Explain Differences between Enzyme-Linked Immunosorbent Assays and Western Blot Assays

2010 ◽  
Vol 17 (11) ◽  
pp. 1739-1745 ◽  
Author(s):  
Lori D. Wendland ◽  
Paul A. Klein ◽  
Elliott R. Jacobson ◽  
Mary B. Brown
Keyword(s):  
Western Blot ◽  
Goodness Of Fit ◽  
False Negative ◽  
Well Being ◽  
Management Decision ◽  
Enzyme Linked Immunosorbent Assay ◽  
Upper Respiratory Tract ◽  
Strain Variation ◽  
Serum Samples ◽  
Mycoplasma Agassizii

ABSTRACT The precarious status of desert (Gopherus agassizii) and gopher (G. polyphemus) tortoises has resulted in conservation efforts that now include health assessment as an important component of management decision-making. Mycoplasmal upper respiratory tract disease (URTD) is one of very few diseases in chelonians for which comprehensive and rigorously validated diagnostic tests exist. In this study, serum samples obtained from eight Gopherus tortoises documented at necropsy to (i) be enzyme-linked immunosorbent assay (ELISA) seropositive using the PS6 antigen, (ii) be infected with Mycoplasma agassizii as indicated by direct isolation of the pathogen from the respiratory surfaces, and (iii) have histological lesions of mycoplasmal URTD were used to evaluate four distinct clinical isolates of M. agassizii as antigens for ELISA and Western blot analyses. Each animal sample reacted in the Western blot with its homologous M. agassizii strain, but recognition of heterologous M. agassizii strains was variable. Further, individual animals varied significantly with respect to the specific proteins recognized by the humoral immune response. An additional 114 Gopherus serum samples were evaluated using ELISA antigens prepared from the four distinct M. agassizii strains; A 405 values were significantly correlated (r 2 goodness of fit range, 0.708 to 0.771; P < 0.0001) for all antigens tested. The results confirm that strain variation is responsible for the observed differences between Western blot binding patterns. Thus, reliance on a single M. agassizii strain as an antigen in Western blot assays may provide false-negative results. This could have adverse consequences for the well-being of these environmentally sensitive hosts if false-negative animals were relocated to sites consisting of true-negative populations.

scholarly journals Differentiation of Larva Migrans Caused by Baylisascaris procyonis and Toxocara Species by Western Blotting

2009 ◽  
Vol 16 (11) ◽  
pp. 1563-1568 ◽  
Author(s):  
Sriveny Dangoudoubiyam ◽  
Kevin R. Kazacos
Keyword(s):  
Western Blotting ◽  
Cross Reactivity ◽  
High Reactivity ◽  
Larva Migrans ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Human Patient ◽  
Western Blots ◽  
Baylisascaris Procyonis ◽  
Very High

ABSTRACT Baylisascaris procyonis and Toxocara species are two important causes of larva migrans in humans. Larva migrans caused by Toxocara spp. is well known and is diagnosed serologically by enzyme immunoassay. Over a dozen cases of larva migrans and associated eosinophilic encephalitis caused by B. procyonis have also been reported, and at least a dozen additional cases are known. An enzyme-linked immunosorbent assay (ELISA) using the excretory-secretory (ES) antigen of B. procyonis larvae is currently being used in our laboratory as an aid in the diagnosis of this infection in humans. Clinically affected individuals show very high reactivity (measured as the optical density) on this ELISA; however, a one-way cross-reactivity with Toxocara spp. has been observed. As an approach to differentiate these two infections based on serology, we performed Western blots, wherein the B. procyonis ES antigen was reacted with serum samples from individuals known to be positive for either Toxocara spp. or B. procyonis larva migrans. Western blot results showed that B. procyonis antigens of between 30 and 45 kDa were specifically identified only by the sera from individuals with Baylisascaris larva migrans, thus allowing for differentiation between the two infections. This included human patient serum samples submitted for serologic testing, as well as sera from rabbits experimentally infected with B. procyonis. When used in conjunction with the ELISA, Western blotting could be an efficient tool for diagnosis of this infection in humans.

scholarly journals Studies on the specific immunodiagnosis of cystic echinococcosis in camels using enzyme-linked immunosorbent assay

2019 ◽  
Vol 22 (3) ◽  
pp. 305-313 ◽  
Author(s):  
O. Kandil ◽  
N. Hassan ◽  
D. Sedky ◽  
E. Beshir Ata
Keyword(s):  
Cystic Echinococcosis ◽  
Haemonchus Contortus ◽  
Indirect Elisa ◽  
Cross Reactivity ◽  
Economic Losses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hydatid Cysts ◽  
Germinal Layer ◽  
Lung Cysts ◽  
Fasciola Spp

Cystic echinococcosis (CE) is of increasing public health and socio-economic concern because of the large morbidity rates and produced high economic losses in the livestock industry. The objective of the current research was to study the reliability of indirect ELISA in detecting CE, based on two dif-ferent types of crude antigens of camel origin; protoscolex and germinal layer antigens from hydatid cyst. Blood samples were collected from 284 (125 slaughtered and 159 live camels). Out of 125 slaughtered camels examined visually, 55 (44%) were found to have hydatid cysts. Of them, 52/125 (41.6%) and 3/125 (2.4%) harboured hydatid cysts in lungs and livers respectively. Fertile lung cysts were 32.8%; 26.9% were sterile, while 40.3% of lung and liver cysts were calcified. The sensitivity of ELISA was 83% and 46.5% when protoscolex and germinal layer antigens were used, respectively. The respective specificity of antigens of protoscolex and germinal layer was 70.3% and 41.7%. The protoscolex antigen showed higher accuracy (73.6%) compared to the germinal layer antigen (52.8%). The cross reactivity of these antigens were evaluated with antigens and hyperimmune sera of CE and Fasciola spp. and Haemonchus contortus using ELISA. The results showed also weak immunogenic potency of each antigen with Fasciola spp. hyperimmune sera at dilution 1:50 while hyperimmune sera of Haemonchus contortus did not bind any antigen.

scholarly journals Development and Potential Application of Ras Domain Containing Protein from Haemonchus contortus for Diagnosis of Goat Infection

Animals ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 138 ◽  
Author(s):  
Kalibixiati Aimulajiang ◽  
Man Cao ◽  
Shuyi Liao ◽  
Muhammad Ali-ul-Husnain Naqvi ◽  
Xiaowei Tian ◽  
...  
Keyword(s):  
Haemonchus Contortus ◽  
Indirect Elisa ◽  
Fasciola Hepatica ◽  
Trichinella Spiralis ◽  
Clinical Signs ◽  
Small Ruminants ◽  
Gastrointestinal Nematode ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fecal Examination ◽  
Diagnostic Potential

Haemonchus contortus is an important gastrointestinal nematode of small ruminants that causes significant mortality in goats worldwide. Diagnosis of this infection mainly depends on the evaluation of clinical signs and fecal examination. However, limitations often occur in early or mild infections. For this purpose, serological diagnosis seems to be more accurate and reliable. Ras domain-containing protein (Ras) is one of H. contortus’s excretory and secretory products (ESPs) that can be isolated from different larval stages of the nematode. In this study, the recombinant H. contortus Ras domain-containing protein (rHcRas) was expressed and purified and its diagnostic potential was evaluated. Reactions between rHcRas and goat sera were tested using Western blotting (WB). The results showed that rHcRas could be recognized by sera as early as 14 days post infection (DPI), and antibodies against rHcRas in infected goats could be maintained for over 89 days. No reaction was found between rHcRas and antibodies against Trichinella spiralis, Fasciola hepatica, or Toxoplasma gondii. An indirect enzyme-linked immunosorbent assay (ELISA) was produced based on rHcRas. The optimal coating antigen (157 ng of rHcRas/well) and serum dilutions (1:50) were determined via checkerboard titration. Indirect ELISA based on rHcRas showed 87.5% sensitivity and 90.6% specificity. The cut-off values for this experiment were determined to be 0.324 (positive) and 0.273 (negative), respectively, and the variation coefficient (CV) was less than 15%. The results of the indirect ELISA in-field examination showed that 17.6% (9/51) of the goats were infected with H. contortus, higher than the fecal examination results (15.7%, 8/51). When compared the results of the indirect ELISA and necropsy testing, 98.0% (50/51) consistency was found. These results indicated that rHcRas was a potential antigen for the diagnosis of H. contortus infection in goats.

scholarly journals Performance of a 27 kDa Fasciola hepatica Antigen in the Diagnosis of Human Fascioliasis

2015 ◽  
Vol 7 (01) ◽  
pp. 017-020 ◽  
Author(s):  
Reza Shafiei ◽  
Bahador Sarkari ◽  
Seyed Mahmoud Sadjjadi
Keyword(s):  
Sensitivity And Specificity ◽  
Western Blotting ◽  
Indirect Elisa ◽  
Fasciola Hepatica ◽  
Serological Diagnosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Healthy Controls ◽  
Elisa System ◽  
Immunodominant Antigen ◽  
Human Fascioliasis

ABSTRACT Background: Serological diagnosis, based on antigenic fractions of the parasite can be used for the early diagnosis of human fascioliasis. The current study aimed to evaluate the efficacy of a 27 kDa immunodominant antigen of Fasciola hepatica adult worms, in an indirect enzyme-linked immunosorbent assay (ELISA) system for serological diagnosis of human fascioliasis. Materials and Methods: The immunodiagnosis of human fascioliasis, using a 27 kDa immunodominant antigen, purified from F. hepatica somatic antigens (SAs), was evaluated by Western blotting and ELISA with sera samples of human fascioliasis patients, healthy controls and patients with other parasitic infections. Results: Using western blotting, from 12 sera of fascioliasis patients, 11 sera (91.6%) detected the 27 kDa subunit. None of 30 samples from healthy controls or 32 sera from nonfascioliasis patients reacted with the 27 kDa antigen. Accordingly, sensitivity and specificity of the system was found to be 91.6% and 100%, respectively. The 27 kDa antigen was purified from the SAs and was used in an indirect ELISA system. Of 15 sera of fascioliasis patients, all (100%) were found to be positive by ELISA whereas only 4 cases (6.25%) of nonfascioliasis patients or healthy controls were false-positive by this system. Accordingly, the sensitivity and specificity of the test were 100% and 93.6%, respectively. Conclusion: Findings of this study demonstrated that both Western blotting and the indirect ELISA, based on the 27 kDa subunit of F. hepatica SA, are reliable methods for serodiagnosis of human fascioliasis.

scholarly journals Cross-Reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique

1997 ◽  
Vol 39 (5) ◽  
pp. 253-256 ◽  
Author(s):  
Cáris Maroni NUNES ◽  
Regina Nardini TUNDISI ◽  
José Fernando GARCIA ◽  
Marcos Brayan HEINEMANN ◽  
Saemi OGASSAWARA ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Western Blotting ◽  
Ascaris Suum ◽  
Toxocara Canis ◽  
Cross Reactivity ◽  
Clinical Syndrome ◽  
Larva Migrans ◽  
Enzyme Linked Immunosorbent Assay ◽  
Visceral Larva Migrans ◽  
Serum Samples

Visceral larva migrans (VLM) is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA) using the larval excretory-secretory antigen of T. canis (TES), the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenicaly related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa). Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis e A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed

scholarly journals Dogs as Sentinels for Flavivirus Exposure in Urban, Peri-Urban and Rural Hanoi, Vietnam

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 507
Author(s):  
Long Pham-Thanh ◽  
Thang Nguyen-Tien ◽  
Ulf Magnusson ◽  
Vuong Bui-Nghia ◽  
Anh Bui-Ngoc ◽  
...  
Keyword(s):  
Mosquito Control ◽  
Risk Mitigation ◽  
Significant Risk ◽  
Cross Sectional Study ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Cross Sectional ◽  
Major Health ◽  
Household Level

Diseases caused by flaviviruses, including dengue fever and Japanese encephalitis, are major health problems in Vietnam. This cross-sectional study explored the feasibility of domestic dogs as sentinels to better understand risks of mosquito-borne diseases in Hanoi city. A total of 475 dogs serum samples from 221 households in six districts of Hanoi were analyzed by a competitive enzyme-linked immunosorbent assay (cELISA) for antibodies to the pr-E protein of West Nile virus and other flaviviruses due to cross-reactivity. The overall flavivirus seroprevalence in the dog population was 70.7% (95% CI = 66.4–74.8%). At the animal level, significant associations between seropositive dogs and district location, age, breed and keeping practice were determined. At the household level, the major risk factors were rural and peri-urban locations, presence of pigs, coil burning and households without mosquito-borne disease experience (p < 0.05). Mosquito control by using larvicides or electric traps could lower seropositivity, but other measures did not contribute to significant risk mitigation of flavivirus exposure in dogs. These results will support better control of mosquito-borne diseases in Hanoi, and they indicate that dogs can be used as sentinels for flavivirus exposure.

scholarly journals Development of a Smartphone-Based Nanozyme-Linked Immunosorbent Assay for Quantitative Detection of SARS-CoV-2 Nucleocapsid Phosphoprotein in Blood

2021 ◽  
Vol 12 ◽  
Author(s):  
Bochao Liu ◽  
Ze Wu ◽  
Chaolan Liang ◽  
Jinhui Lu ◽  
Jinfeng Li ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Cross Reactivity ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Primary Screening ◽  
Global Pandemic ◽  
Novel Coronavirus ◽  
Acid Test ◽  
Control Samples

Since December 2019, a novel coronavirus (SARS-CoV-2) has resulted in a global pandemic of coronavirus disease (COVID-19). Although viral nucleic acid test (NAT) has been applied predominantly to detect SARS-CoV-2 RNA for confirmation diagnosis of COVID-19, an urgent need for alternative, rapid, and sensitive immunoassays is required for primary screening of virus. In this study, we developed a smartphone-based nanozyme-linked immunosorbent assay (SP-NLISA) for detecting the specific nucleocapsid phosphoprotein (NP) of SARS-CoV-2 in 37 serum samples from 20 COVID-19 patients who were diagnosed by NAT previously. By using SP-NLISA, 28/37 (75.7%) serum samples were detected for NP antigens and no cross-reactivity with blood donors’ control samples collected from different areas of China. In a control assay using the conventional enzyme-linked immunosorbent assay (ELISA), only 7/37 (18.91%) serum samples were detected for NP antigens and no cross-reactivity with control samples. SP-NLISA could be used for rapid detection of SARS-CoV-2 NP antigen in primary screening of SARS-CoV-2 infected individuals.

Sign in / Sign up

Export Citation Format

Share Document