Direct Detection Of Viral Antigens Research Articles

An ultrasensitive fluorescence aptasensor for SARS-CoV-2 antigen based on hyperbranched rolling circle amplification

Sensitive and accurate diagnosis of SARS-CoV-2 infection at early stages can help to attenuate the effects of the COVID-19. Compared to RNA and antibodies detection, direct detection of viral antigens could reflect infectivity more appropriately. However, it is still a great challenge to construct a convenient, accurate and sensitive biosensor with a suitable molecular recognition element for SARS-CoV-2 antigens. Herein, we report a HRCA-based aptasensor for simple, ultrasensitive and quantitative detection of SARS-CoV-2 S1 protein and pseudovirus. The aptamer sequence used here is selected from several published aptamers by enzyme-linked oligonucleotide assay and molecular docking simulation. The sensor forms an antibody-target-aptamer sandwich complex on the surface of microplates and elicits HRCA for fluorescent detection. Without complicated operations or special instruments and reagents, the aptasensor can detect S1 protein with a LOD of 89.7 fg/mL in the linear range of 100 fg/mL to 1 μg/mL. And it can also detect SARS-CoV-2 spike pseudovirus in artificial saliva with a LOD of 51 TU/μL. Therefore, this simple and ultrasensitive aptasensor has the potential to detect SARS-CoV-2 infection at early stages. It may improve the timeliness and accuracy of SARS-CoV-2 diagnosis and demonstrate a strategy to conduct aptasensors for other targets.

Immunodominant regions prediction of nucleocapsid protein for SARS-CoV-2 early diagnosis: a bioinformatics and immunoinformatics study

ABSTRACT COVID-19 caused by SARS-CoV-2 is sweeping the world and posing serious health problems. Rapid and accurate detection along with timely isolation is the key to control the epidemic. Nucleic acid test and antibody-detection have been applied in the diagnosis of COVID-19, while both have their limitations. Comparatively, direct detection of viral antigens in clinical specimens is highly valuable for the early diagnosis of SARS-CoV-2. The nucleocapsid (N) protein is one of the predominantly expressed proteins with high immunogenicity during the early stages of infection. Here, we applied multiple bioinformatics servers to forecast the potential immunodominant regions derived from the N protein of SARS-CoV-2. Since the high homology of N protein between SARS-CoV-2 and SARS-CoV, we attempted to leverage existing SARS-CoV immunological studies to develop SARS-CoV-2 diagnostic antibodies. Finally, N229-269, N349-399, and N405-419 were predicted to be the potential immunodominant regions, which contain both predicted linear B-cell epitopes and murine MHC class II binding epitopes. These three regions exhibited good surface accessibility and hydrophilicity. All were forecasted to be non-allergen and non-toxic. The final construct was built based on the bioinformatics analysis, which could help to develop an antigen-capture system for the early diagnosis of SARS-CoV-2.

A novel biosensor based on serum antibody immobilization for rapid detection of viral antigens

In this paper, we represent a label-free biosensor based on immobilization of serum antibodies for rapid detection of viral antigens. Human serum containing specific antibodies against Japanese encephalitis virus (JEV) was immobilized on a silanized surface of an interdigitated sensor via protein A/glutaraldehyde for electrical detection of JEV antigens. The effective immobilization of serum antibodies on the sensor surface was verified by Fourier transform infrared spectrometry and fluorescence microscopy. The signal of the biosensor obtained by the differential voltage converted from the change into non-Faradic impedance resulting from the specific binding of JEV antigens on the surface of the sensor. The detection analyzed indicates that the detection range of this biosensor is 1–10μg/ml JEV antigens, with a detection limit of 0.75μg/ml and that stable signals are measured in about 20min. This study presents a useful biosensor with a high selectivity for rapid and simple detection of JEV antigens, and it also proposes the biosensor as a future diagnostic tool for rapid and direct detection of viral antigens in clinical samples for preliminary pathogenic screenings in the case of possible outbreaks.

Transgenic cell lines for detection of animal viruses.

Rapid diagnostic assays based on direct detection of viral antigen or nucleic acid are being used with increasing frequency in clinical virology laboratories. Virus culture, however, remains the only way to detect infectious virus and to analyze clinically relevant viral phenotypes, such as drug resistance. Growth of viruses in cell culture is labor intensive and time-consuming and requires the use of many different cell lines. Transgenic technology, together with increasing knowledge of the molecular pathways of virus replication, offers the possibility of using genetically modified cell lines to improve virus growth in cell culture and to facilitate detection of virus-infected cells. Genetically modifying cells so that they express a reporter gene only after infection with a specific virus can allow the detection of infectious virus by rapid and simple enzyme assays such as beta-galactosidase assays without the need for antibodies. Although transgenic cells have recently been successfully used for herpes simplex virus detection, much more work needs to be done to adapt this technology to other human viral pathogens such as cytomegalovirus and respiratory viruses. This review offers some strategies for applying this technology to a wide spectrum of animal viruses.

AIDS testing in the 1990s

The principal screening tests for HIV infection are serologic assays for viral-specific antibody. The most commonly used tests are either enzyme immunoassays or enzyme-linked immunosorbent assays. The sensitivity of these tests is approximately 98%; the specificity is almost 100%. The Western blot is presently the definitive confirmatory test for HIV infection. To be considered positive, the assay must show reactivity to three specific viral proteins: p24 (“gag”), p31 (“pol”), and either gp41, gp120, or gp160 (“env”). The specificity of the Western blot is essentially 100%. Other diagnostic tests for HIV infection include direct detection of viral antigen by polymerase chain reaction and viral culture. Virus is cultured most readily from peripheral blood lymphocytes and monocytes. These latter methods are not available as readily and are not as inexpensive as antibody detection assays and, thus, are not widely used in daily clinical practice.

Transgenic cell lines for detection of animal viruses.

Rapid diagnostic assays based on direct detection of viral antigen or nucleic acid are being used with increasing frequency in clinical virology laboratories. Virus culture, however, remains the only way to detect infectious virus and to analyze clinically relevant viral phenotypes, such as drug resistance. Growth of viruses in cell culture is labor intensive and time-consuming and requires the use of many different cell lines. Transgenic technology, together with increasing knowledge of the molecular pathways of virus replication, offers the possibility of using genetically modified cell lines to improve virus growth in cell culture and to facilitate detection of virus-infected cells. Genetically modifying cells so that they express a reporter gene only after infection with a specific virus can allow the detection of infectious virus by rapid and simple enzyme assays such as beta-galactosidase assays without the need for antibodies. Although transgenic cells have recently been successfully used for herpes simplex virus detection, much more work needs to be done to adapt this technology to other human viral pathogens such as cytomegalovirus and respiratory viruses. This review offers some strategies for applying this technology to a wide spectrum of animal viruses.

Rapid and sensitive streptavidin‐biotin amplified fluorogenic enzyme‐linked immunosorbent‐assay for direct detection and identification of dengue viral antigens in serum

Each of the four serotypes of dengue viruses is responsible for a spectrum of illnesses that range from nonspecific febrile syndrome with good prognosis to dengue haemorrhagic fever or dengue shock syndrome. Definite diagnosis of dengue is provided by the detection of virus in acute-phase sera of patients. Virus isolation can be accomplished with mosquito cell lines or mosquito inoculations. However, these methods are time consuming and labour intensive. The reverse-transcriptase polymerase chain reaction (RT-PCR) provides a potential means of rapid diagnosis but requires specialised facilities and equipment and is expensive. Therefore a rapid, simple, sensitive, and economical method for direct detection of viral antigens in viraemic sera is needed for clinical and epidemiological investigations. An amplified fluorogenic enzyme-linked immunosorbent assay (F-ELISA) is described for the detection and identification of dengue-3 viruses in serum specimens. This assay utilizes biotinylated mouse IgG antibody directed against dengue antigens captured by anti-dengue monoclonal antibody coated onto polystyrene microplate wells. It takes advantage of the high affinity of biotin for the multivalent binding sites of streptavidin-labelled beta-galactosidase, and combines the amplification effect of biotin-streptavidin interaction with the high sensitivity of fluorogenic detection methods. Following optimisation of the procedure by reducing non-specific binding of proteins and enhancing the specific binding of antigens, F-ELISA was tested on 259 sera submitted routinely to our laboratory for confirmation of dengue diagnosis. The sensitivity of the F-ELISA was 90%, the specificity was 99% and the agreement rate was 98% between F-ELISA and virus isolation results.

Measles encephalitis during immunosuppressive treatment for acute lymphoblastic leukaemia.

Between 1971 and 1989 measles encephalitis was identified in five children receiving chemotherapy for acute lymphoblastic leukaemia. Review of these and previously reported cases of measles encephalitis in immunosuppressed patients failed to identify any pathognomonic features in the history, the clinical presentation, or the results of electroencephalography or computed tomography. Detection of measles virus antigen in nasopharyngeal secretions or intrathecal synthesis of specific antibody was not possible in all instances. Early diagnosis by direct detection of viral antigen in the brain was confounded by difficulties in identifying areas of the brain suitable for biopsy. Increasing herd immunity to measles in the general population by vaccination is the only effective intervention against measles encephalitis in immunosuppressed children. Measles encephalitis must be remembered as a possible explanation of encephalopathy in the immunocompromised child: the benefits of early use of antiviral agents need to be evaluated.

Vertical transmission of avian adenovirus associated with inclusion body hepatitis

Using an indirect enzyme-linked immunsorbent assay, avian adenoviral antigens were detected in the yolk and albumen of eggs derived from broiler breeder chickens which were known to be infected with a strain of virus capable of causing inclusion body hepatitis. Viral antigens were detected in egg yolk (16/60) more frequently than in the albumen (5/60). Direct detection of viral antigens in eggs strongly supports the hypothesis that transovarian transmission of inclusion body hepatitis virus occurs if infection is present in breeder flocks.

Mixed infection is common in children with respiratory adenovirus infection.

ABSTRACT. The presence of concomitant viral or bacterial infection was evaluated in 20 patients hospitalized for adenovirus infection of the middle or lower airways by using new serological methods for detection of both antigens and antibodies. Adenovirus infection was identified by measurement of antibodies with complement fixation test or by direct detection of viral antigen in nasopharyngeal aspirates. Mixed infection was present in 11 (55%) of the 20 patients. Viral coinfection was demonstrated in five (25%) and bacterial in nine (45%) patients. Bacterial coinfection was common, 67%, in children with an infection focus, pneumonia or acute otitis media, but rare, 13%, in those without it. Seroconversion to nontypable Haemophilus influenzae was indicated in six children; four of them were infants, four had pneumonia and three acute otitis media. Pneumococcal infection was indicated in two patients with pneumonia, both aged over two years. Chlamydia trachomatis was involved in one case. The results indicate that bacterial coinfection is common in respiratory adenovirus infection affecting lower airways, especially if pneumonia is present.

Generation and application of a monoclonal antibody raised against a recombinant cytomegalovirus-specific polypeptide

Procedures for diagnostics of cytomegalovirus infections include histopathology, cell culture, serology, and direct detection of viral antigens or nucleic acids within infected cells or tissues. In order to develop a new diagnostic reagent for viral antigen detection, we generated a mouse monoclonal antibody. This antibody was raised against a recombinant antigen representing part of the large phosphorylated structural protein pp150 of human cytomegalovirus. The monoclonal antibody was shown to be useful for antigen detection by immunofluorescence and immunoenzymatic staining in infected cells from cell culture as well as from infected organs. The antibody proved to be reactive even in paraffin-embedded sections from tissue specimens.

Rapid detection of respiratory syncytial virus and influenza A virus in cell cultures by immunoperoxidase staining with monoclonal antibodies

Peroxidase-labeled monoclonal antibodies against respiratory syncytial virus (RSV) and influenza A virus were used for immunoperoxidase staining (IPS) of cell cultures inoculated with nasopharyngeal aspirates. Cells were grown in 24-well plates, and specimens were inoculated by low-speed centrifugation. Cultures were incubated for 2 days at 37 degrees C and then fixed, stained, and observed by light microscopy. IPS was compared with standard virus isolation by using cultures of human diploid fibroblasts and Vero, HEp-2, and HeLa cell lines for RSV and Madin-Darby canine kidney cells for influenza A virus; these cultures were inoculated with specimens that were previously stored at -70 degrees C. Of 40 known RSV-positive specimens, 30 were found to be positive on reinoculation by both methods, and an additional 5 specimens were found to be positive by IPS only. Of 190 specimens tested for influenza A virus, 14 were positive by IPS and in tubes, and a further 8 specimens were positive by IPS only. IPS was also compared with direct detection of viral antigens in nasopharyngeal aspirates by a time-resolved fluoroimmunoassay (TR-FIA). Fresh nasopharyngeal aspirates were inoculated into human diploid fibroblasts and Madin-Darby canine kidney cells and tested for RSV and influenza A virus, respectively, by IPS. Of 110 specimens tested for RSV, 37 were positive in total, 32 were positive by IPS, and 33 were positive by TR-FIA. Of 150 specimens tested for influenza A virus, 39 were positive in total, 35 were positive by IPS, and 34 were positive by TR-FIA. IPS of cultures inoculated by centrifugation and incubated for 2 days is a sensitive method for the diagnosis of respiratory virus infections, and 24-well plates allow for the easy processing of a large number of specimens.

Production of polyclonal antisera against cytomegalovirus and varicella‐zoster virus by use of affinity‐purified viral antigens

Specific animal antisera to cytomegalovirus (CMV) and varicella-zoster virus (VZV), devoid of antibodies to host cell components, have been difficult to prepare because of the cell-associated nature of these viruses. A simple method for the purification of CMV and VZV antigens is described in this paper. The viral antigens were purified from commercially available crude antigens by affinity chromatography with human antibodies against CMV and VZV. Rabbits were immunized with the purified antigens. The produced antisera were specific for the respective viruses and did not contain antibodies to host cell components, as determined by immunofluorescence microscopy and enzyme-immunoassay. The antisera have been used for direct detection of viral antigens in clinical specimens and for identification of cytopathic effect in cell cultures by immunofluorescence microscopy. The CMV antiserum could also be used for the early detection of CMV in cell cultures inoculated with patient specimens, but it was less sensitive than a monoclonal antibody to the early antigen of CMV.

Rapid diagnosis of rift valley fever: A comparison of methods for the direct detection of viral antigen in human sera

Human sera collected during the 1987 Rift Valley fever (RVF) epidemic in the Senegal River basin were analysed using three enzyme immunoassays to establish the best method for rapid diagnosis of RVF. A biotin-avidin-enhanced antigen detection method utilizing monoclonal antibodies proved most sensitive. Eighty-two viremic human sera were tested, and this assay detected antigen in 29.3% of the samples.

Viral findings in children under the age of two years with expiratory difficulties.

ABSTRACT. Viral findings were prospectively studied in lower respiratory tract infections in small children with and without expiratory difficulties. On first admission, a viral aetiology was found in 71 of 127 children (56%). On re‐admission, a viral etiology was found in only two of 31 cases (6%). Respiratory syncytial viruses (RSV) were responsible for 71% of the cases with viral diagnoses. A recently‐developed method for the direct detection of viral antigens in nasopharyngeal specimens by radio‐immunoassay was more sensitive than complement fixation serology, especially in patients aged less than six months. Viral diagnosis was reached using this new method alone in 43% of infections caused by RSV and in 27% of infections caused by other viruses. In children under six months, RSV were found in 89% by direct antigen detection and in 22% by serology. We suggest that direct antigen detection should be used as the primary virological method in small children with lower respiratory tract infections. The aetiological agents were the same in cases with and without expiratory difficulties, RSV being found in about 40% of children in both instances. It is concluded that host factors are critical to the development of expiratory difficulties.

Diagnosis of respiratory syncytial virus infection in children: comparison of viral antigen detection and serology.

Direct detection of viral antigen in nasopharyngeal secretion by radioimmunoassay was compared with serology by IgG antibody enzyme immunoassay for diagnostic efficacy in 77 children with clinically suspected respiratory syncytial virus (RSV) infections. Antigen detection gave a positive diagnosis in 26 of 33 (79%) children in whom RSV infection was diagnosed by any of the two methods. The diagnostic efficacy of antigen detection was dependent upon the interval after onset at which specimens were collected; 88% of specimens taken during the first 5 days and 50% of specimens taken 6-10 days after onset of illness were positive. It was also dependent on the age of the patients, the diagnostic efficacy being 88 and 76% in children under and over 6 months of age, respectively.

Immunoassay diagnosis of adenovirus infections in children.

Direct detection of viral antigen in nasopharyngeal secretions and stool specimens by radioimmunoassay and the determination of serum antibody responses by complement fixation and immunoglobulin class-specific enzyme immunoassay against the hexon antigen were compared for diagnostic efficacy in 52 children with acute adenovirus infections. The highest diagnosis rate (85% of the cases) was obtained by antigen detection in nasopharyngeal secretions. Adenovirus antigen was also detected in stools of 72% of the 18 patients tested. The immunoglobulin G (IgG) antibody enzyme immunoassay detected 77% of the cases, being more sensitive than the complement fixation test with a 67% detection rate. The IgM antibody response was variable with no clear correlation with the age of the patient or severity of the clinical symptoms. IgM antibody response was detected in 48% of the patients and had the normal transient course, with a persistence of the IgM antibodies of approximately 2 months. Determination of IgA antibodies gave a diagnostic increase in titer in 37% of the cases and was found less suitable for serological diagnosis. Because of the clinical importance of rapid laboratory diagnosis, the direct detection of viral antigen in nasopharyngeal secretions or stool or both should be used as the primary diagnostic test in adenovirus infections.

Sensitive enzyme immunoassay with beta-D-galactosidase-Fab conjugate for detection of type A influenza virus antigen in clinical specimens.

The most sensitive method for diagnosis of type A influenza virus infection is isolation of the agent in cell culture. However, detection and identification may require several days to complete. This delay in diagnosis prevents effective use of the antiviral agents available for treatment of type A influenza infection. As a rapid diagnostic method, enzyme immunoassay (EIA) is attaining increased usage for direct detection of viral antigen in clinical specimens. Standard EIA techniques, however, are usually not sensitive enough for reliable detection of viral antigen in respiratory secretions. We developed a conjugate consisting of the antigen-binding fragment of goat antirabbit immunoglobulin G coupled to beta-d-galactosidase, using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Other immunoreagents in our EIA consisted of guinea pig and rabbit antisera to influenza A/Brazil/11/78 (H1N1) for microtiter plate coating and primary antiserum, respectively. The sensitivity of this EIA was tested with 60 clinical specimens containing influenza A/England/333/80 (H1N1) which closely resembles A/Brazil. Of 31 initial specimens, collected within 24 h of the onset of symptoms, 27 (87%) were positive, using a fluorgenic substrate, and 18 of 29 (62%) specimens obtained 12 to 60 h after the initial specimens were positive, for a total of 75% (45 of 60). All positive reactions were specific, as shown in a confirmatory test with preimmune and hyperimmune guinea pig globulins. Clinical specimens negative for virus (n = 33) or containing heterologous respiratory viruses (n = 26) were negative in this system. These results indicate that EIA systems can be developed with a sensitivity approaching that required for clinical usefulness.