Antigen Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Reston Ebola Virus Nucleoprotein

Antigen capture enzyme-linked immunosorbent assay (ELISA) is one of the most useful methods to detect Ebola virus rapidly. We previously developed an antigen capture ELISA using a monoclonal antibody (MAb), 3-3D, which reacted not only to the nucleoprotein (NP) of Zaire Ebola virus (EBO-Z) but also to the NPs of Sudan (EBO-S) and Reston Ebola (EBO-R) viruses. In this study, we developed antigen capture ELISAs using two novel MAbs, Res2-6C8 and Res2-1D8, specific to the NP of EBO-R. Res2-6C8 and Res2-1D8 recognized epitopes consisting of 4 and 8 amino acid residues, respectively, near the C-terminal region of the EBO-R NP. The antigen capture ELISAs using these two MAbs detected the EBO-R NP in the tissues from EBO-R-infected cynomolgus macaques. The antigen capture ELISAs using Res2-6C8 and Res2-1D8 are useful for the rapid detection of the NP in EBO-R-infected cynomolgus macaques.     

Enzyme-Linked Immunosorbent Assay Method for Detection of Cytomegalovirus Strain-Specific Antibody Responses

Reliable methods for the detection of cytomegalovirus (CMV) strain-specific serological responses are lacking. We describe a simple and reliable enzyme-linked immunosorbent assay method developed to detect antibodies against the polymorphic epitopes within the two envelope glycoproteins of CMV, glycoproteins H and B. This assay is useful for the detection of serologic responses to CMV strains and the identification of CMV reinfections.Cytomegalovirus (CMV) is an important pathogen in immunocompromised hosts and a frequent cause of congenital infection. CMV isolated from clinical samples exhibits extensive genetic variation (7, 12, 13), and CMV reinfections have been demonstrated to occur in seropositive individuals. However, it is thought that these reinfections have little untoward consequences with respect to congenital infections. Recent studies documenting higher rates of congenital CMV infection in populations with nearly universal seroreactivity to CMV suggest that infection with new or different virus strains could be responsible for the intrauterine transmission of CMV in immune mothers (5, 17, 18). The frequency and consequences of infection with multiple CMV strains are unclear because of the lack of reliable methods for the accurate identification of CMV strain-specific antibody responses. By utilizing the defined heterogeneity within the antibody binding epitopes on envelope glycoprotein H (gH) and gB of the AD169 and Towne strains of CMV, an enzyme-linked immunosorbent assay (ELISA) method was developed to distinguish serological responses against infection with different CMV strains.Serum samples from 96 CMV-seropositive women participating in an ongoing study and 51 seronegative individuals were tested for anti-CMV strain-specific antibodies. Informed consent was obtained from the study participants, and the study was conducted in accordance with the guidelines of the Institutional Review Board for Human Use of the University of Alabama at Birmingham.Purified recombinant antigens based on polymorphic antibody binding sites defined on gH (antigen gpUL75) and gB (antigen gpUL55) were used as antigens (Fig. ​(Fig.1).1). The gH antigens were constructed as β-galactosidase fusion proteins containing the coding region for amino acids (aa) 15 to 142 of gpUL75 from the AD169 strain (the AP86 antigen) and aa 14 to 42 of the Towne strain (the TO86 antigen) of CMV (16). The recombinant peptides were expressed in Escherichia coli and were purified as described previously (8). gB antigens were prepared as six-His-tag-labeled peptides by cloning the coding region (aa 1 to 116) from strains AD169 (the AD55 antigen) and Towne (the TO55 antigen) (9) into expression vector pET21a (EMD, Gibbstown, NJ) by using the HindIII and BamHI endonuclease restriction sites. The peptides were expressed in E. coli Rosetta cells and were purified by using Talon Superflow metal affinity columns (Clonetech, Mountain View, CA). A positive control antigen was constructed by cloning the antigen domain 1 (AD-1) region of the gene coding gB, which has been shown to be highly conserved among clinical isolates of CMV, as described previously (2), into each vector (AD-1). The reactivity against an empty vector expressing fusion protein alone or nonantigenic proteins of mouse origin was used as a negative control.Open in a separate windowFIG. 1.Amino acid sequence alignment of the amino-terminal regions of gpUL75 (gH) and gpUL55 (gB) from the AD169 and Towne strains of CMV depicting the differences betweens the two strains.Strain-specific ELISA was performed on PolySorp microtiter plates (Nunc, Roskilde, Denmark). The wells of the plates were coated overnight with 50 μl of purified gH antigens (antigens AP86 and TO86) (1) or gB antigens (antigens AD55 and TO55) diluted in carbonate buffer and blocked with 3% goat serum in borate buffer (BB) for 2 h at 37°C. Serum samples diluted 1:100 in BB were added to the wells, and the plates were incubated at 37°C for 1 h. After the plates were washed three times with BB containing 0.05% Tween 20, goat anti-human immunoglobulin G (IgG) horseradish peroxidase (HRP)-conjugated antibody (Pierce, Rockford, IL) diluted 1:10,000 was added and the plates were incubated for 1 h at 37°C. The plates were developed by the addition of 50 μl of one-step Ultra TMB (3,3′,5,5′-tetramethylbenzidine) substrate (Pierce) for 10 min at room temperature (RT), and the reaction was stopped by the addition of 2 N sulfuric acid. The optical density (OD) values were determined with a spectrophotometer. A positive result was defined as an OD value more than three times the mean result obtained for each antigen with seronegative samples.Western blot assays were performed with a subset of 12 samples. Appropriate antigens were run on a 12.5% sodium dodecyl sulfate-polyacrylamide gel and then blotted onto a polyvinylidene difluoride membrane (Immobilon P; Millipore, Billerica, MA), according to the manufacturer''s recommendations. The membranes were blocked for 2 h in 3% goat serum in SuperBlock buffer (Pierce, Rockford, IL) and 0.05% Tween 20 at RT. Human sera were diluted 1:5,000 in blocking buffer and applied to the membrane, and the membrane was shaken at RT for 2 h. The membranes were washed four times in wash buffer (BB with 0.05% Tween 20), and goat anti-human IgG HRP-conjugated antibody (Pierce) diluted 1:100,000 in blocking buffer was added. After incubation at RT for 2 h, the membranes were washed four times in wash buffer and soaked into the substrate Luminol West Femmto (Pierce) for 10 min. The membranes were placed on X-ray film, and images were developed and acquired by using a VersaDoc imaging system (Bio-Rad, Hercules, CA).Of the 96 baseline serum samples from CMV-seropositive women participating in an ongoing study testing for strain-specific antibodies, 58 (60%) samples were positive for at least one of the four antigens and 18 samples were positive for two or more antigens. The OD values (mean ± standard deviation) for each antigen for the group of 51 CMV-seronegative individuals and the samples that were considered positive and negative from the 96 CMV IgG antibody-positive women are shown in the Table ​Table1.1. Forty-five percent (43/96) of the samples had reactivity to the AP86 antigen, with a mean OD value of 1.240 ± 0.498, whereas the OD values were 0.291 ± 0.134 and 0.196 ± 0.052 for the negative samples and the CMV IgG antibody-negative control serum samples, respectively. Fifteen percent (14/96) of the study samples were positive for the TO86 antigen, with an average OD reading of 1.069 ± 0.317. Responses against the AD55 and TO55 antigens were seen in 8% and 20% of the samples, respectively, with corresponding OD values of 0.681 ± 0.103 and 0.708 ± 0.278 (Table ​(Table11).

TABLE 1.

Reactivities against the gH (AP86 and TO86) and gB (AD55 and TO55) polymorphic epitopes from CMV strains AD169 and Towne of serum samples from 96 CMV-seropositive women and 51 seronegative individuals and two serum samples with known reactivity against AP86, TO55, or TO86

Evaluation and Diagnostic Usefulness of Domestic and Imported Enzyme-Linked Immunosorbent Assays for Detection of Human Immunodeficiency Virus Type 1 Antibody in India

Diagnosis of human immunodeficiency virus (HIV) infection is important for patient management and prevention of new infections. The number of test kits available for the detection of HIV antibodies is unprecedented. In order to identify appropriate test kits, we evaluated a variety of commercial kits manufactured abroad as well as in India. The plasma and serum specimens (n = 264) were collected from individuals attending the Voluntary Counseling and Testing Centre at the YRG Centre for AIDS and Education. The specimens were used to evaluate six commercially available HIV test kits: Enzaids HIV 1+2, HIV-CheX, Murex HIV-1.2.0, Genscreen HIV 1/2 version 2, Vironostika HIV Uni-Form II Ag/Ab, and CombAids RS Advantage. High sensitivities and specificities (≥99%) were observed for the Enzaids, Murex, Vironostika, and CombAids assays. HIV-CheX showed the highest number of false-positive and false-negative results. The Genscreen test also gave many false positives. The study indicated that the Enzaids, Murex, and Vironostika enzyme-linked immunosorbent assay kits and the CombAids RS Advantage rapid assay could be used to achieve acceptable results for the detection of HIV antibodies. A combination of two tests is recommended to optimize the efficiency of HIV antibody testing algorithms, especially when evaluation with an HIV Western blot confirmatory test is not possible.     

Development and Applications of a Bovine Coronavirus Antigen Detection Enzyme-Linked Immunosorbent Assay

We developed a monoclonal antibody-based, antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) for bovine coronavirus. We compared the ELISA with electron microscopy and the hemagglutination test and found a close correlation between them. The sensitivity of the ELISA was 10⁴ bovine coronavirus particles per ml of 10% fecal suspension. Compared with electron microscopy, bovine coronavirus ELISA had 96% specificity.     

Serological Evaluation of Thin-Layer Immunoassay–Enzyme-Linked Immunosorbent Assay for Antibody Detection in Human Trichinellosis

A new immunoenzymatic test, named the thin-layer immunoassay–enzyme-linked immunosorbent assay (TIA-ELISA), was evaluated for antibody detection in human trichinellosis using excretion and secretion products prepared from Trichinella spiralis muscle larvae. Serum samples from people with positive muscle biopsies or symptoms compatible with the disease (n = 8 or 26, respectively), all reactive in enzyme-linked immunoelectrotransfer blot assay (EITB), as well as 67 serum samples from healthy, EITB-negative people, were tested in an ELISA and TIA-ELISA. TIA-ELISA was performed in polystyrene plastic petri dishes by adding dots of 10 μl each of antigen (7 μg/ml) followed by adding diluted serum and the conjugate. Finally, the substrate mixed with agar was added to develop the reaction. Enzymatic by-products were easily detected by the naked eye as defined dots. Sensitivity and specificity were 76 and 94% for ELISA, and both parameters were 91% for TIA-ELISA. The kappa correlation indices for both tests in relation to EITB were 0.73 and 0.80, respectively. The TIA-ELISA can be carried out with common laboratory equipment in 3 h and uses lower quantities of antigen than EITB and ELISA. Since TIA-ELISA is easy to perform, cheap, sensitive, and specific, the test could be an acceptable alternative to use in clinical laboratories lacking specialized equipment needed for ELISA and EITB and in field studies for antibody detection in human trichinellosis.     

Noninfectious Virus-Like Particle Antigen for Detection of Swine Vesicular Disease Virus Antibodies in Pigs by Enzyme-Linked Immunosorbent Assay

An inactivated SVDV antigen is used in current enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to swine vesicular disease virus (SVDV). To develop a noninfectious recombinant alternative, we produced SVDV-like particles (VLPs) morphologically and antigenically resembling authentic SVDV particles by using a dual baculovirus recombinant, which expresses simultaneously the P1 and 3CD protein genes of SVDV under different promoters. Antigenic differences between recombinant VLPs and SVDV particles were not statistically significant in results obtained with a 5B7-ELISA kit, indicating that the VLPs could be used in the place of SVDV antigen in ELISA kits. We developed a blocking ELISA using the VLPs and SVDV-specific neutralizing monoclonal antibody 3H10 (VLP-ELISA) for detection of SVDV serum antibodies in pigs. The VLP-ELISA showed a high specificity of 99.9% when tested with pig sera that are negative for SVDV neutralization (n = 1,041). When tested using sera (n = 186) collected periodically from pigs (n = 19) with experimental infection with each of three different strains of SVDV, the VLP-ELISA detected SVDV serum antibodies as early as 3 days postinfection and continued to detect the antibodies from all infected pigs until termination of the experiments (up to 121 days postinfection). This test performance was similar to that of the gold standard virus neutralization test and indicates that the VLP-ELISA is a highly specific and sensitive method for the detection of SVDV serum antibodies in pigs. This is the first report of the production and diagnostic application of recombinant VLPs of SVDV. Further potential uses of the VLPs are discussed.     

Cloning of the Bovine Immunodeficiency Virus gag Gene and Development of a Recombinant-Protein-Based Enzyme-Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific bovine immunodeficiency virus (BIV) antibodies in cattle, using recombinant Gag protein as an antigen. The gag coding region from BIV was cloned into an expression vector, pQE32, which expressed high levels of recombinant protein from Escherichia coli. The ELISA was standardized by a checkerboard titration against known BIV-positive and -negative sera from cattle and a monoclonal antibody to the Gag protein. A total of 139 cattle serum samples, from the diagnostic laboratory at Kansas State University, Manhattan, and from the Dairy Station, Louisiana State University, Baton Rouge, were compared by ELISA and immunoblot assays for the detection of BIV-specific antibodies. Of 26 cattle sera samples which tested positive using the immunoblot assay, 23 were positive by ELISA, thus establishing a strong correlation between the two tests. The sensitivity and specificity of ELISA relative to immunoblotting were 0.88 and 0.93, respectively. ELISA proved to be as specific as immunoblotting but was much less time-consuming and easier to perform.     

Lack of Correlation Between the Immunobead Test and the Enzyme-Linked Immunosorbent Assay for Sperm Antibody Detection

ABSTRACT: A total of 41 sera submitted for routine sperm antibody screening were tested by both the indirect immunobead test (IBT) against viable sperm as antigen, and an enzyme-linked immunosorbent assay (ELISA) using the supernatant fraction from washed, sonicated sperm as antigen. Sixteen sera were positive by either IBT or ELISA, but none were positive by both tests. It was concluded that the tests may be detecting antibodies reacting with distinct antigen subgroups, possibly because the ELISA may be detecting antibodies directed against internal antigens or non-sperm-specific antigens such as HLA.     

Detection of Infectious Pancreatic Necrosis Virus Using an Inhibition Enzyme-Linked Immunosorbent Assay

Abstract

An inhibition enzyme-linked immunosorbent assay was used to detect infectious pancreatic necrosis (IPN) virus. In this assay the presence of virus was determined by measuring the decrease in titer of a known antiserum after incubation with a sample suspected to contain virus. The titer of the antiserum was measured with an indirect enzyme-linked assay. Compared to the double antibody sandwich method this assay required fewer reagents (only one anti- IPN serum was required). This assay was also sensitive enough to detect virus at levels of 1×10² TCID₅₀/ml. of purified virus and was able to detect virus in samples obtained in the field.     

LcrV Capture Enzyme-Linked Immunosorbent Assay for Detection of Yersinia pestis from Human Samples

In the United States, there is currently a major gap in the diagnostic capabilities with regard to plague. To address this, we developed an antigen capture assay using an essential virulence factor secreted by Yersinia spp., LcrV, as the target antigen. We generated anti-LcrV monoclonal antibodies (MAbs) and screened them for the ability to bind bacterially secreted native Yersinia pestis LcrV. Anti-LcrV MAb 19.31 was used as a capture antibody, and biotinylated MAb 40.1 was used for detection. The detection limit of this highly sensitive Yersinia LcrV capture enzyme-linked immunosorbent assay is 0.1 ng/ml. The assay detected LcrV from human sputum and blood samples treated with concentrations as low as 0.5 ng/ml of bacterially secreted native Y. pestis LcrV. This assay could be used as a tool to help confirm the diagnosis of plague in patients presenting with pneumonia.     

Heterophile Antibodies to Bovine and Caprine Proteins Causing False-Positive Human Immunodeficiency Virus Type 1 and Other Enzyme-Linked Immunosorbent Assay Results

Heterophile antibodies are a well-recognized cause of erroneous results in immunoassays. We describe here a 22-month-old child with heterophile antibodies reactive with bovine serum albumin and caprine proteins causing false-positive results to human immunodeficiency virus type 1 and other infectious serology testing.     

Comparison of Seven Commercial Antigen and Antibody Enzyme-Linked Immunosorbent Assays for Detection of Acute Dengue Infection

Seven commercial assays were evaluated to determine their suitability for the diagnosis of acute dengue infection: (i) the Panbio dengue virus Pan-E NS1 early enzyme-linked immunosorbent assay (ELISA), second generation (Alere, Australia); (ii) the Panbio dengue virus IgM capture ELISA (Alere, Australia); (iii) the Panbio dengue virus IgG capture ELISA (Alere, Australia); (iv) the Standard Diagnostics dengue virus NS1 antigen ELISA (Standard Diagnostics, South Korea); (v) the Standard Diagnostics dengue virus IgM ELISA (Standard Diagnostics, South Korea); (vi) the Standard Diagnostics dengue virus IgG ELISA (Standard Diagnostics, South Korea); and (vii) the Platelia NS1 antigen ELISA (Bio-Rad, France). Samples from 239 Thai patients confirmed to be dengue virus positive and 98 Sri Lankan patients negative for dengue virus infection were tested. The sensitivities and specificities of the NS1 antigen ELISAs ranged from 45 to 57% and 93 to 100% and those of the IgM antibody ELISAs ranged from 85 to 89% and 88 to 100%, respectively. Combining the NS1 antigen and IgM antibody results from the Standard Diagnostics ELISAs gave the best compromise between sensitivity and specificity (87 and 96%, respectively), as well as providing the best sensitivity for patients presenting at different times after fever onset. The Panbio IgG capture ELISA correctly classified 67% of secondary dengue infection cases. This study provides strong evidence of the value of combining dengue virus antigen- and antibody-based test results in the ELISA format for the diagnosis of acute dengue infection.     

Detection of Antibody against Extractable Nuclear Antigen by an Enzyme-Linked Immuno-Sorbent Assay

Anti-ENA antibody determination by ELISA technique may offer a valuable diagnostic help in the discrimination of patients with mixed connective tissue disease (MCTD) from those with other chronic inflammatory connective tissue diseases. Determination of this antibody was performed in a prospective designed investigation among 101 blood donors, 154 patients with various non-rheumatic internal medical diseases, and 229 patients with chronic inflammatory connective tissue diseases, including five patients with MCTD. A positive titre of anti-ENA antibody was found in approximately 10% of blood donors and patients with various internal medical disorders. A highly positive anti-ribonucleoprotein (RNP) titre was found in the patients with MCTD, but was also observed in patients with other chronic inflammatory connective tissue diseases, giving a predictive value of 56% for MCTD. We conclude that highly positive anti-RNP antibody values do not automatically indicate the diagnosis MCTD. Other diagnostic possibilities should still be considered.     

Optimization of a Human Papillomavirus-Specific Enzyme-Linked Immunosorbent Assay

A strategy was developed for the control, standardization, and critical evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of human papillomavirus-specific immunoglobulin G in human sera. Control human sera, polyclonal animal sera, and monoclonal antibodies were used to establish optimal assay parameters, including antigen coating, serum dilutions, and criteria for daily reproducibility, monitoring, and rejection of assays. Three evaluation techniques were used in parallel to define an optimal cutoff absorbance value that yields greater than 93% sensitivity and 98.5% specificity in the assay's ability to discriminate positive and negative control sera. This strategy provides an optimal method by which to determine cutoff absorbance values for ELISA.     

Early Detection of Human Immunodeficiency Virus Infection Using Third- and Fourth-Generation Screening Assays

 Early detection of infection with human immunodeficiency virus (HIV) is critical for clinical diagnosis and treatment of patients, as well as for ensuring the safety of blood transfusion products. Recently, a number of fourth-generation HIV screening assays have been developed that offer increased sensitivity over earlier tests by combining detection of anti-HIV antibodies with detection of the p24 viral antigen. Previously, six different HIV assays were compared against a broad range of 30 seroconversion panels. In the present study, three of the newer fourth-generation assays were tested together with three of the third-generation HIV antibody-only assays. This extensive analysis highlights (i) the importance of p24 antigen detection for early diagnosis, (ii) the improved sensitivity of fourth-generation assays over antibody-only tests, and (iii) the superior performance of the Vidas Duo assay, which allows reduction of the diagnostic window by up to 2 weeks. Finally, the results emphasize the detection limitations of the different assays and suggest improvements for future HIV screening assays.     

Development of Two Antibody Detection Enzyme-Linked Immunosorbent Assays for Serodiagnosis of Human Chronic Fascioliasis

Coprological examination based on egg detection in stool samples is currently used as the gold standard for the diagnosis of human fascioliasis. However, this method is not effective during the acute phase of the disease and has poor sensitivity during the chronic phase. Serodiagnosis has become an excellent alternative to coprological examination in efforts to combat the effects of fascioliasis on human and animal health. Two novel recombinant Fasciola hepatica proteins, i.e., a ferritin (FhFtn-1) and a tegument-associated protein (FhTP16.5), were used as antigens to develop in-house enzyme-linked immunosorbent assay (ELISA) methods. The assays were optimized and validated using 152 serum samples from humans with a known infection status, including healthy subjects, patients with chronic fascioliasis, and patients with other parasitic diseases. The FhFtn-1 ELISA was shown to be 96.6% sensitive and 95.7% specific; the respective parameters for the FhTP16.5 ELISA were 91.4% and 92.4%. The performances of the FhFtn-1 and FhTP16.5 ELISAs were compared with that of an available commercial test (the DRG test) using a subset of serum samples. Our in-house tests were slightly more sensitive than the DRG test in detecting antibodies against F. hepatica, but the differences were not statistically significant. In conclusion, the present study provides evidence for the potential of the FhFtn-1 and FhTP16.5 ELISAs as diagnostic tools for human fascioliasis, as might be implemented in conjunction with standard assays for large-scale screenings in areas where the disease is endemic and for the detection of occasional cases in clinical laboratories.     

ELISA检测妇女生殖器疱疹中单纯疱疹病毒型特异性抗原的研究

用抗HSV型共同性McAb和抗HSV-2型特异性McAb包被微板,建立了能检测妇女生殖器疤疹的宫颈或阴道棉拭标本中HSV抗原,并可分型的ELISA。经与病毒分离和中和试验分型对照研究,证明本法快速、敏感、特异,分型准确可靠,可用于大规模现场普查。用本法分型检测了宫颈糜烂、霉菌性阴道炎、淋病和滴虫性阴道炎标本中的HSV抗原,结果提示上述疾病均可感染或并发感染HSV,其中以HSV-2感染为主,占24.51％,HSV-1感染次之,占6.94％。宫颈糜烂的程度与HSV阳性检出率呈正相关。淋病患者并发HSV感染的机会更多,其中HSV-1感染较其它疾病多见。故预防HSV性传播和产道感染给新生儿,应得到高度重视。