Development of a fibrinogen-specific sandwich enzyme-linked immunosorbent assay microarray assay for distinguishing between blood plasma and serum samples

Feline sera from Bursa province (Turkey) were assayed for coronavirus antibody using an enzyme-linked immunosorbent assay (ELISA). The study was performed on 100 sera collected from cats belonging to catteries or community shelters and to households. The serum samples were initially tested with the virus neutralisation (VN) test and the results were then compared with the ELISA. The VN yielded 79 negative and 21 positive sera but the ELISA confirmed only 74 as negative. The ELISA-negative sera were also found to be free of feline coronoviruses-specific antibodies by Western blotting. Using the VN as the gold standard test, ELISA had a sensitivity of 100% and a specificity of 93.6%, with an overall agreement of 95%. The Kappa (κ) test indicated high association between the two tests (κ=0.86, 95% confidence interval (CI) 0.743–0.980). The positive predictive value (PPV) was 0.8, and the negative predictive value (NPV) was 0.93. The prevalence of FCoV II antibodies in the sampled population based on the gold standard was 62% (95% CI 0.44–0.77) among multi-cat environments, and 4% (95% CI 0.01–0.11) among single cat households.

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Development of an Enzyme-Linked Immunosorbent Assay for the Detection of Antibody to Epizootic Hemorrhagic Disease of Deer Virus

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870001200207 ◽

2000 ◽

Vol 12 (2) ◽

pp. 142-145 ◽

Cited By ~ 18

Author(s):

James O. Mecham ◽

Michael M. Jochim

Keyword(s):

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Hemorrhagic Disease ◽

Structural Protein ◽

Serum Samples ◽

Epizootic Hemorrhagic Disease ◽

Diagnostic Potential ◽

The Us ◽

Serotype 1 ◽

Infected Cells

An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1-and EHDV-2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo.

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

Frontiers in Microbiology ◽

10.3389/fmicb.2021.692831 ◽

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.

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Development of an Enzyme-Linked Immunosorbent Assay for Immunoglobulin M Antibodies against Measles Virus

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.10.3.439-442.2003 ◽

2003 ◽

Vol 10 (3) ◽

pp. 439-442 ◽

Cited By ~ 3

Author(s):

F. Roodbari ◽

M. H. Roustai ◽

A. Mostafaie ◽

H. Soleimanjdahi ◽

R. Sarrami Foroshani ◽

...

Keyword(s):

Immunosorbent Assay ◽

Neutralizing Antibodies ◽

Measles Virus ◽

Clinical Symptoms ◽

Maculopapular Rash ◽

Immunoglobulin M ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Vaccination Programs ◽

Elisa System

ABSTRACT Measles is a highly contagious respiratory virus infection, with typical clinical symptoms including maculopapular rash, fever, cough, coryza, and conjunctivitis. Despite implementation of widespread vaccination programs throughout the world, the rates of global morbidity and mortality are still considerable. This study was performed to design a reliable indirect enzyme-linked immunosorbent assay (ELISA) to measure measles-specific immunoglobulin M (IgM). First, human IgM was purified, and then an anti-IgM antibody was produced in rabbits and purified in a multistep process. The rabbit IgG against human IgM was conjugated with peroxidase. Measles virus-infected Vero cells produced viral antigen. One hundred serum samples from infants of 9 to 18 months of age, mostly vaccinated, were evaluated for determining the presence of specific IgM antibodies against measles virus. The samples were also evaluated for neutralizing antibodies against measles virus by a microneutralization test (MNT). By comparing the results of the ELISA with those of MNT, it was demonstrated that ELISA had a sensitivity and specificity of 100 and 92%, respectively. On the other hand, when the results obtained by our ELISA system were compared with those of an imported measles virus IgM ELISA kit (EIAgen; Adaltis Italia SPa, Bologna, Italy), a high level of agreement was shown (k = 0.926).

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An enzyme-linked immunosorbent assay for the detection of IgG antibodies against Babesia equi in horses

Ciência Rural ◽

10.1590/s0103-84782004000500031 ◽

2004 ◽

Vol 34 (5) ◽

pp. 1525-1529 ◽

Cited By ~ 12

Author(s):

Cristiane Divan Baldani ◽

Rosangela Zacarias Machado ◽

Paulo de Tarso Landgraf Botteon ◽

Felipe Santoro Takakura ◽

Carlos Luiz Massard

Keyword(s):

Immunosorbent Assay ◽

Serological Test ◽

Epidemiological Studies ◽

Sao Paulo ◽

Enzyme Linked Immunosorbent Assay ◽

São Paulo ◽

Igg Antibodies ◽

Serum Samples ◽

Babesia Equi ◽

Specificity And Sensitivity

A crude antigenic preparation of Babesia equi was used to develop and establish the suitability of an enzyme-linked immunosorbent assay (ELISA) for the detection of parasite carriers. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkboard titrations. The specificity and sensitivity of the ELISA were 100 %. A total of 90 serum samples were taken from horses from the Northeast region of São Paulo State and examined for diagnosis of equine B. equi infection by ELISA. Approximately 75% (n=67) of all the horses tested were found serologically positive for B. equi. These results suggest that the ELISA described may prove to be an appropriate serological test for epidemiological studies on B. equi infections in the field and that equine piroplasmosis is a cause for serious concern in the State of São Paulo, Brazil.

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Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia

Journal of Clinical Microbiology ◽

10.1128/jcm.03259-15 ◽

2016 ◽

Vol 54 (6) ◽

pp. 1557-1565 ◽

Cited By ~ 1

Author(s):

Martin Heller ◽

Nimmo Gicheru ◽

Georgina Tjipura-Zaire ◽

Cecilia Muriuki ◽

Mingyan Yu ◽

...

Keyword(s):

Immunosorbent Assay ◽

Rapid Test ◽

Lateral Flow ◽

Sub Saharan Africa ◽

Enzyme Linked Immunosorbent Assay ◽

Contagious Bovine Pleuropneumonia ◽

Serum Samples ◽

Content Type ◽

Mycoplasma Mycoides ◽

Sub Saharan

Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease that is widespread in sub-Saharan Africa. It is caused byMycoplasma mycoidessubsp.mycoides, a bacterium belonging to theMycoplasma mycoidescluster. In the absence of an efficient CBPP vaccine, improved and easy-to-use diagnostic assays for recurrent testing combined with isolation and treatment of positive animals represent an option for CBPP control in Africa. Here we describe the comprehensive screening of 17 immunogenicMycoplasma mycoidessubsp.mycoidesproteins using well-characterized bovine sera for the development of a novel cocktail enzyme-linked immunosorbent assay (ELISA) for laboratory use. Two recombinantMycoplasmaimmunogens, MSC_0136 and MSC_0636, were used to set up a standardized cocktail ELISA protocol. According to the results from more than 100 serum samples tested, the sensitivity and specificity of the novel cocktail ELISA were 85.6% and 96.4%, respectively, with an overall diagnostic accuracy comparable to that of the Office International des Epizooties (OIE)-prescribed serological assays. In addition, we provide a proof of principle for a field-applicable, easy-to-use commercially produced prototype lateral-flow test for rapid (<30-min) diagnosis of CBPP.

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Evaluation of an Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Orientia tsutsugamushi Infection

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.10.3.394-398.2003 ◽

2003 ◽

Vol 10 (3) ◽

pp. 394-398 ◽

Cited By ~ 19

Author(s):

Won-Jong Jang ◽

Myung-Suk Huh ◽

Kyung-Hee Park ◽

Myung-Sik Choi ◽

Ik-Sang Kim

Keyword(s):

Immunosorbent Assay ◽

Scrub Typhus ◽

Microtiter Plate ◽

Immunoglobulin M ◽

Enzyme Linked Immunosorbent Assay ◽

Orientia Tsutsugamushi ◽

Serum Samples ◽

Capture Elisa ◽

Igm Antibodies ◽

Immunodominant Antigen

ABSTRACT To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We developed an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) for diagnosis of recent Orientia tsutsugamushi infections in humans. The 56-kDa major outer membrane protein of O. tsutsugamushi is well known as the most immunodominant antigen in scrub typhus. The test is based on the use of the biotinylated recombinant 56-kDa protein of O. tsutsugamushi Boryong, Bor56, which was expressed as a fusion protein with a maltose-binding protein in Escherichia coli. In the test, the serum IgM antibodies were captured by anti-human IgM antibodies coated onto a microtiter plate. The captured IgM antibodies were revealed through sequential addition of biotinylated Bor56 antigen and peroxidase-conjugated streptavidin to the plate. The IgM capture ELISA was compared with the immunofluorescence antibody assay (IFA) by testing 176 serum samples from patients with diagnosed cases of rickettsial disease and patients with other acute febrile diseases. Of the 81 IgG IFA-positive samples, 78 tested positive (sensitivity, 96.3%) and all 31 IgM IFA-positive samples tested positive (sensitivity, 100%) by the IgM capture ELISA. The specificity of the IgM capture ELISA was 99%, and 1 of the 95 IFA-negative samples was positive in the assay. These results strongly suggest that IgM capture ELISA using the recombinant Bor56 antigen is a reliable and detailed method for the detection of early O. tsutsugamushi infection.

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Enzyme-Linked Immunosorbent Assay for Detection of Plasmodium falciparum Histidine-Rich Protein 2 in Blood, Plasma, and Serum

Clinical and Vaccine Immunology ◽

10.1128/cvi.00385-07 ◽

2008 ◽

Vol 15 (6) ◽

pp. 1012-1018 ◽

Cited By ~ 60

Author(s):

Carolyne M. Kifude ◽

Halli G. Rajasekariah ◽

David J. Sullivan ◽

V. Ann Stewart ◽

Evelina Angov ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Immunosorbent Assay ◽

Whole Blood ◽

Linear Range ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Target Concentration ◽

Intervention Trials ◽

Lower Limits ◽

Asexual Cycle

ABSTRACT Microscopy, the gold standard for the detection and quantification of malaria parasites in blood, is in many aspects deficient for this purpose. The method is poorly reproducible and can be inaccurate because Plasmodium falciparum parasites sequester for a portion of each asexual cycle. Due to these deficiencies, biomarkers such as P. falciparum histidine-rich protein 2 (PfHRP2) are increasingly being used. In this study, we evaluated the use of a commercial PfHRP2 enzyme-linked immunosorbent assay (ELISA) kit with some procedural modifications. We determined the linear range of the assay, including the lower limits of detection and quantitation, using recombinant PfHRP2 (rPfHRP2). In 10 repeat experiments, the linear range of optical densities (ODs) at 450 to 650 nm was from 0.05 ± 0.002 to 2.28 ± 0.042, corresponding to 3.91 to 250 ng/ml of rPfHRP2. The coefficient of variation (CV) at each target concentration ranged from 1.93 to 8.07%. Using cultured parasites, we confirmed the linear range of ODs as well as the association between the PfHRP2 ELISA results and the microscopic parasite densities. For whole-blood samples spiked with cultured, washed, ring-stage-infected red blood cells (iRBCs), the linear range was 11.7 to 750 iRBCs/μl, with CVs of 0.29 to 7.56%. The same spiked samples evaluated by microscopists had similar sensitivities, but the CVs were unacceptably high (20.7 to 161.6%). Stock rPfHRP2 was stable through four freeze-thaw cycles (P < 0.05; paired t test). When different patient sample types at different concentrations within the linear range of the assay are compared, the recoveries of PfHRP2 from blood and serum were within ±20%, whereas the recoveries from plasma ranged between +35 and −41%. We conclude that PfHRP2 ELISA using whole-blood and serum samples is a suitable adjunct to microscopy and could ultimately benefit malaria intervention trials.

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A study of the correlation between Coxiella burnetii seropositivity and abortions in sheep in Eastern and Southeastern Turkey

Indian Journal of Animal Research ◽

10.18805/ijar.9364 ◽

2016 ◽

Cited By ~ 1

Author(s):

Ayse Kilic ◽

Hakan Kalender

Keyword(s):

Immunosorbent Assay ◽

Coxiella Burnetii ◽

Q Fever ◽

Antibody Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Eastern Anatolia ◽

Obligate Intracellular Bacterium ◽

Serum Samples ◽

Obligate Intracellular ◽

Elisa Kit

Q fever is a zoonotic disease that occurs worldwide and is caused by the obligate intracellular bacterium Coxiella burnetii. Infected animals are usually asymptomatic, but infection can cause abortion and stillbirth in ruminants. The main purpose of this study was to evaluate prevalance of Coxiella burnetii infection in aborted and nonaborted sheep serum samples in Eastern Anatolia region by using enzyme-linked immunosorbent assay (ELISA). The determine of prevalance in sheep flocks from four provinces (Elazig, Malatya, Tunceli, Bitlis) and tested for anti-C.burnetii antibody detection, by means of Chekit Q fever Elisa kit. 350 serum samples obtained from flocks belonging aborted sheep showed that a total of 56 (16%) were detected seropositivity, whereas 171 serum samples obtained from nonaborted sheep flocks in 13 of the 171 (7.60%) for C.burnetii in seropositivity were observed. Coxiellosis should be considered an important cause of sheep with abortion history and nonaborted in Elazig and neighboring provinces.

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