Determination of herpes simplex virus type-specific antibodies by enzyme-linked immunosorbent assay.

We determined type-specific antibodies to herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) by an indirect enzyme-linked immunosorbent assay, using as antigens HSV-1 glycoprotein gC-1 and a HSV-2-specific polypeptide purified on affinity columns of monoclonal antibodies. All sera were initially screened for HSV antibodies by the enzyme-linked immunosorbent assay with a pool of Triton X-100-extracted antigens of HSV-1- and HSV-2-infected HEp-2 cells. The titer of HSV antibodies was predicted from a linear regression curve based on the absorbance of the initial 1:50 serum dilution. The sensitivity and specificity of the screening assay and of the assay for type-specific antibodies were established.     

Typing of herpes simplex virus by capture biotin-streptavidin enzyme-linked immunosorbent assay and comparison with restriction endonuclease analysis and immunofluorescence method using monoclonal antibodies.

A sensitive enzyme-linked immunosorbent capture assay using biotin and streptavidin (capture B/SA ELISA) was developed using type-specific monoclonal antibodies for typing of herpes simplex virus. Rabbit anti-herpes simplex virus immunoglobulin G was used as the capturing antibody, and biotin-linked type-1-specific mouse monoclonal antibody or rabbit type-1- or type-2-specific polyclonal antibody served as the detecting antibody. The captured antigen was detected by an ELISA with alkaline phosphatase-conjugated streptavidin, which reacted with biotin molecules on the detector antibody. The capture B/SA ELISA was compared with other methods for efficiency and reliability in typing. Results obtained by restriction endonuclease digestion of the radiolabeled viral genome were used to determine the type (1 or 2) of clinical isolates. These results were then used as a reference for determining the accuracy of the capture B/SA ELISA, as well as that of the immunofluorescence method, both of which are easily adaptable for use in the clinical laboratory. The three methods were in perfect agreement. It was determined that both the capture B/SA ELISA and the immunofluorescence method using monoclonal antibodies provided typing results with 100% specificity and 100% sensitivity and thus were accurate and reliable. However, the ELISA was the method of choice because of its simplicity, rapidity, and use of nonradioisotopic reagents.     

Detection of herpes simplex virus type-specific antibodies by an enzyme-linked immunosorbent assay based on glycoprotein G

In order to develop a simple and quantitative method to detect herpes simplex virus (HSV) type-specific antibodies, the usefulness of an enzyme-linked immunosorbent assay (ELISA) using HSV glycoprotein G (gG) captured on a plate by monoclonal antibodies as antigen was studied. The gG1- and gG2-specific IgG antibody activities were measured by the ELISA for 54 sera which had been collected from culture-proven genital herpes patients and pre-characterized by an immunodot assay using purified gG antigens. Thirty control sera without antibodies against the HSV whole antigens were also included. In comparison with the immunodot assay as standard, the sensitivities of the ELISA were 88.9% (32/36) for HSV-1 antibody and 89.2% (33/37) for HSV-2 antibody and the specificities were both 100%. Sera taken within a few months after primary infection tended to give false negative results. The HSV type-specific ELISA based on easy-to-prepare gG antigens might be useful to help improve the serological assessment of HSV infections. J. Med. Virol. 53:319–323, 1997. © 1997 Wiley-Liss, Inc.     

Rapid diagnosis of herpes simplex virus infections by enzyme-linked immunosorbent assay.

A total of 457 clinical specimens were tested for the presence of herpes simplex virus (HSV) by isolation in cell culture and by enzyme-linked immunosorbent assay. HSV antigen was detected by enzyme-linked immunosorbent assay in 94% of the clinical specimens from which the virus was isolated. The detection rate was improved to 100% when specimens were either assayed within 24 h or stored frozen at a constant temperature (-20 degrees C) for up to 14 days. Type-specific HSV antigen was also detected in 6% of culture-negative specimens. The assay can be completed in 5 h or less, and commercially available immunoglobulins are used. Enzyme-linked immunosorbent assay is a rapid and sensitive method for the diagnosis of HSV infection and can be used to improve the management of pregnant women with a history of genital herpes and of neonates and others with serious HSV infection which may require specific antiviral therapy. It also offers an alternative to cell culture for routine diagnosis of HSV infections.     

Detection of herpes simplex virus in clinical specimens by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) for the detection of herpes simplex virus is described that can be performed in approximately four hours. The test, which does not require specialized equipment and uses relatively inexpensive, commercially available reagents, detected herpes simplex virus in 51% of specimens found to be positive by a time-consuming cell culture technic. The ELISA test compared favorably with a direct immunofluorescence method that detected HSV in only 1% of the cell culture-positive specimens. The ELISA test was readily carried out even with specimens unsuitable for cell culture and did not require cellular material as is the case with immunofluorescence technics. An advantage of the ELISA test for herpes simplex virus over the cell culture method was the detection of nonviable virus.     

Typing of herpes simplex virus isolates by enzyme-linked immunosorbent assay: comparison between indirect and double-antibody sandwich techniques.

Indirect and double-antibody sandwich enzyme-linked immunosorbent assay techniques were compared for serotyping 42 herpes simplex virus isolates. Both procedures gave similar results, which were in complete agreement with those obtained by inhibition of the indirect hemagglutination test. The indirect technique proved to be as specific as the double-antibody sandwich enzyme-linked immunosorbent assay, but simpler and cheaper.     

Evaluation of solubilized herpes simplex virus membrane antigen by enzyme-linked immunosorbent assay

An antigen prepared by solubilization of membranes from herpes simplex virus (HSV)-infected cells with deoxycholate was evaluated by enzyme-linked immunosorbent assay. The deoxycholate-solubilized antigen, previously shown to contain all major HSV glycoproteins, was noninfectious and adsorbed easily and reproducibly to a polystyrene surface at pH 9.6. The deoxycholate-solubilized antigen provided an enzyme-linked immunosorbent assay of high sensitivity and reproducibility with complete correlation with complement fixation for the diagnosis of acute HSV infection. The correlation with neutralization and immunofluorescence for the presence or absence of anti-HSV activity was very good. Comparison with an HSV envelope preparation yielded results slightly in favor of the deoxycholate-solubilized antigen. The assay seems to be useful for demonstration of intrathecal production of antibody activity in HSV encephalitis.     

Evaluation of an enzyme-linked immunosorbent assay for the detection of herpes simplex virus antigen.

An enzyme-linked immunosorbent assay (ELISA) kit for herpes simplex virus developed by Ortho Diagnostic Systems, Inc., was evaluated. In phase I experiments, 263 clinical specimens from genital lesions were extracted into serum-free medium and then tested by ELISA for herpes simplex virus antigen. The results were compared with those obtained by conventional viral culture. Of 83 specimens, 65 were positive by ELISA (sensitivity, 78.3%). In phase II experiments, 249 clinical specimens were tested for herpes simplex virus antigen in direct specimen and in cell cultures (MRC-5 and rabbit kidney) incubated for 2, 4, and 7 days. Of 63 specimens, 40 were positive by ELISA in the direct specimen (sensitivity, 63.5%), and by 7 days incubation, 100% of the cultures positive by viral cell culture were also positive by ELISA. The ELISA was reproducible, and when both the direct detection and amplification culture were used, the sensitivity of ELISA paralleled the diagnosis of herpes simplex virus infections by viral cytopathic effect.     

Serotyping herpes simplex virus isolated by enzyme-linked immunosorbent assays.

An enzyme-linked immunosorbent assay (ELISA) using a double-antibody sandwich tecnhique has been developed to serotype isolates of herpes simplex virus from clinical sources. The results obtained using this procedure were in agreement with those obtained with a standard neutralization test in typing stock cultures and 32 clinical isolates of herpes simplex virus. Clear differentiation between the two viral serotypes was obtained using rabbit immunoglobulin cross-absorbed with heterologous virus antigen. The ELISA procedure described appears to be a convenient and accurate substitute for the neutralization test in typing herpes simplex viruses. ELISA techniques require relatively small amounts of antigen and antibody and can be performed with very simple equipment.     

Factors influencing the sensitivity of herpes simplex virus detection in clinical specimens in a simultaneous enzyme-linked immunosorbent assay using monoclonal antibodies

A rapid simultaneous enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was investigated for herpes simplex virus (HSV) detection. All HSV isolated (n = 127) were detected, whereas no response was obtained with HSV negative preparations. Equivalent results were obtained from 275 of 277 clinical specimens in the monoclonal ELISA and in an ELISA using polyclonal antibodies, confirming that appropriately selected monoclonal antibodies may be as efficacious as polyclonal antibodies in antibody-based assays. In clinical specimens, the rate of HSV detection (sensitivity) relative to tissue culture isolation was low for both assays, and the major factor responsible for this was the low concentration of virus present in some specimens. The sensitivity of ELISA obtained in routine use varied with different panels of unselected specimens and was related to the speed of development of the cytopathic effect. These results emphasise the need for caution in assigning a definitive sensitivity level to ELISA tests evaluated on different panels of specimens.     

Typing of herpes simplex virus isolates with monoclonal antibodies and by nucleic acid spot hybridization

Fifty-one clinical isolates of herpes simplex virus (HSV) were typed by an enzyme immunoassay (EIA) using mouse monoclonal antibodies, by DNA spot hybridization, and by restriction enzyme analysis using restriction endonuclease Eco RI. Extracts of VERO cells infected with the isolates were used for coating microtitre plates or denatured and spotted onto nitrocellulose filters. Viral antigens passively adsorbed to microtitre plates were detected by an indirect EIA using mouse monoclonal antibodies specific for HSV type 1 (HSV-1) or HSV type 2 (HSV-2). Spotted DNA was hybridized with 32P-labeled probes containing Hind III/Sal I-fragments of either HSV-1 or HSV-2 DNA and bound radioactivity was detected by autoradiography and counted in a liquid scintillation counter. All the three methods gave identical results for the 51 isolates studied. Twenty-six isolates were identified as HSV-1 and 25 as HSV-2. An additional 30 specimens were tested only by EIA and hybridization. Results by both techniques were in complete agreement.     

Detection of influenza virus neuraminidase-specific antibodies by an enzyme-linked immunosorbent assay.

We analyzed sera from 28 patients with various types of malignancies for the occurrence of antibodies against exotoxin A of Pseudomonas aeruginosa and two Pseudomonas proteases. A total of 27 of these individuals were colonized or infected with P. aeruginosa at one time or another during the study, whereas the remaining patient was colonized with four non-P. aeruginosa species of Pseudomonas. Sera were obtained from several of these patients before P. aeruginosa colonization or infection of these individuals was detected, which provided an opportunity to evaluate their responsiveness to pseudomonal exoproducts as they acquired the organism. Exotoxin A was purified from culture supernatant fluids of strain PA-103, and the two proteases were purified from an isolate of strain JR3, a highly proteolytic strain originally recovered from the sputum of a cystic fibrosis patient. Antibodies to the exotoxin A and the two proteases were detected in these sera, and sera which contained relatively high antibody levels to exotoxin A afforded mice complete protection against lethal challenges with this substance. Statistical analyses showed that patients infected with P. aeruginosa had consistently higher antibody levels (P less than 0.005) to the exoproducts than patients who were colonized with this organism. Also, patients colonized with P. aeruginosa possessed significantly higher antibody levels (P less than 0.003) to these three exoproducts than uninfected, hospitalized patients. Parke-Davis type 1 was the strain most commonly isolated from these patients (46%), but colonization or infection due to this organism usually resulted in the production of low levels of antibody to Pseudomonas exoproducts. However, infections with Parke-David type 7 organisms were always associated with intermediate- and high-responder sera to exotoxin A. These results indicated that potentially toxic products were elaborated during the course of cancer-related colonization and infection with P. aeruginosa.     

Comparison of enzyme-linked immunosorbent assay with enzyme-linked fluorescence assay with automated readers for detection of rubella virus antibody and herpes simplex virus.

The enzyme-linked immunosorbent assay (ELISA) was compared with the enzyme-linked fluorescence assay (ELFA) for the detection of rubella antibody and herpes simplex virus antigen. Test parameters, specimens, antigen or antibody, and conjugates for the two types of assays were identical except that p-nitrophenyl phosphate was used as the substrate for the ELISA and 4-methylumbelliferyl phosphate was used as the substrate for ELFA. Automated readers were used for both assays. Antibody titers and sensitivity of antigen detection were quite similar for ELISA and ELFA. ELFA for rubella antibody, however, could be conducted with less antigen or shorter substrate incubation time (5 min for ELFA versus 30 min for ELISA). For herpes simplex virus antigen detection, ELFA could also be read after a shorter substrate incubation time (15 min for ELFA versus 30 min for ELISA). Clear polystyrene microtiter plates routinely used for ELISA could be used for ELFA, but clear polyvinyl chloride plates had high background fluorescence. Black polystyrene and polyvinyl chloride plates gave lower background fluorescence than did clear plates. ELFA is of particular value as a substitute for ELISAs in which long substrate incubations are required or antigens of only low titer are available.     

A sensitive enzyme-linked immunosorbent assay for the detection of herpes simplex virus antigens

A sandwich ELISA for the detection of herpes simplex virus (HSV) antigens was developed using sheep anti-HSV F(ab')2 fragments for capture and an indirect antibody system for detection. Current detection limits are 0.5 ng protein for HSV1 and 1.5 ng protein for HSV2. This compares to a single HSV1-infected Vero-cell in a background of 10(6) non-infected cells or 10 plaque forming units (PFU) of HSV1 in culture supernatants as determined in separate experiments. Limiting dilution experiments show that one PFU of HSV1 can be detected after overnight culture in both supernatant and cell extracts. The use of F(ab')2 for capture completely eliminated binding of Staphylococcus aureus. No cross-reactivity was observed with other human herpes viruses. When evaluated with 245 random 'left-overs' of genital swab specimens in transport medium the test showed a sensitivity and specificity of 77.2 and 97.8%, respectively, with respect to virus isolation in culture. In a preliminary study on 16 direct ELISA swab-specimens extracted in 0.5 ml ELISA sample buffer both sensitivity and specificity were 100% with respect to culture. In both clinical series there was a proportional relationship between the ELISA value and the estimated amount of infectious virus in the specimen.     

Evaluation of a commercial enzyme-linked immunosorbent assay for detection of herpes simplex virus antigen.

A commercial enzyme-linked immunosorbent assay (ELISA) for detection of herpes simplex virus (HSV) antigen in clinical specimens and culture lysates was evaluated. A total of 1,155 specimens and an additional 335 cell culture lysates were tested by ELISA, and results were compared to those obtained by cell culture in primary rabbit kidney, human foreskin, and MRC-5. The sensitivity and specificity of the direct test were 52.5 and 96.9% and those of the culture lysate test were 98.7 and 96.9%, respectively. The sensitivity of the direct ELISA correlated with early cytopathic effect in cell culture and varied with specimen source, ranging from 100% with skin lesions to 40.9% with cervical swabs. Of 60 cervical specimens from asymptomatic individuals, 22 (36.6%) yielded false-positives, which may be due to noninfectious HSV. No reproducible cross-reactions were found with other viruses isolated. The Ortho HSV ELISA was found to be rapid, sensitive, and specific for detection of HSV from cell culture lysates, but it needs reevaluation for direct specimen testing, in particular for screening of asymptomatic obstetrical patients.     

Evaluation of a commercial enzyme-linked immunosorbent assay for the detection of herpes simplex virus.

A total of 136 specimens were tested for the presence of herpes simplex virus by routine tissue culture and a commercial enzyme-linked immunosorbent assay (Ortho Diagnostic Systems, Inc.). Forty-six (33.8%) of the specimens were positive by tissue culture. The sensitivity and specificity of the commercial system were 69.6 and 93.3%, respectively. The commercial system was rapid and moderately specific but lacked the sensitivity necessary for direct specimen testing.     

An enzyme-linked immunosorbent assay for IgG antibodies to human herpes virus 6

An indirect enzyme-linked immunosorbent assay (ELISA) with human herpes virus 6 (HHV6) membrane antigen was compared with indirect immunofluorescence assay (IFA) for measurement of HHV6 IgG antibodies. Five hundred serum samples from 403 Swedish patients with suspected symptomatic Epstein-Barr virus (EBV) infections were examined. The specificity of the ELISA compared with IFA was 98.7% and the sensitivity was 98.4%. In 90% of the patients, IgG antibodies to HHV6 were detected with both assays. The highest HHV6 IgG titers were found mainly in patients with EBV or CMV infections, but HHV6 mononucleosis was not diagnosed. The same HHV6 antigen was assessed for IgM ELISA but was found to be of limited value due to high IgM reactivity with the control antigen. The HHV6 IgM ELISA requires further investigation. The IgG ELISA described is a reliable alternative to IFA for measurement of HHV6 IgG antibodies and for large scale epidemiological studies.     

Identification of serotype 9 human rotavirus by enzyme-linked immunosorbent assay with monoclonal antibodies.

Hybridomas producing monoclonal antibodies to VP7, a major neutralizing protein of serotype 9 rotavirus (strain W161), were prepared. One monoclonal antibody, W161-6A1, was shown to neutralize only serotype 9 rotavirus strains and reacted specifically with serotype 9 rotaviruses in an enzyme-linked immunosorbent assay. The development of an immunoassay for detection of serotype 9 rotaviruses should facilitate epidemiologic studies.     

Detection of human group C rotaviruses by an enzyme-linked immunosorbent assay using monoclonal antibodies.

An enzyme-linked immunosorbent assay using monoclonal antibodies was established for the detection of human group C rotaviruses. Seventeen clinical samples which were found to contain group C rotaviruses were all strongly positive, whereas 9 samples containing group A rotaviruses and 51 samples lacking rotaviruses were all negative with this test.