Detection and typing of herpes simplex viruses by using recombinant immunoglobulin fragments produced in bacteria.

Thirty-seven bacterial clones producing human recombinant monoclonal antibody Fab fragments (rFabs) reactive to herpes simplex virus (HSV) antigens were selected from a human combinatorial antibody library constructed in a phage-display vector by a panning procedure against an HSV lysate. Thirty-four of the HSV-specific rFabs were able to specifically recognize HSV-infected cells in indirect immunofluorescence (IF) assays; of these, 25 recognized cells infected by either HSV type 1 (HSV-1) or HSV-2, while 9 recognized only HSV-1-infected cells. One HSV type-common rFab (rFab H37) and one HSV-1-specific rFab (rFab H85) were further evaluated as reagents for viral detection and typing by IF staining in 134 HSV-positive (72 HSV-1 and 62 HSV-2) viral cultures from clinical specimens. The results obtained with these two rFabs were fully consistent with those obtained with a commercial preparation of fluorescein-labeled anti-HSV type-specific murine monoclonal antibodies. The detection sensitivity with the type-common rFab in indirect IF assays was higher overall than that provided by the type-specific murine monoclonal antibodies. Preparations of rFabs suitable for IF staining can be easily and inexpensively obtained in a clinical microbiology laboratory from Escherichia coli cultures. Similar HSV-specific rFabs, therefore, could be advantageous for in vitro diagnostic purposes.     

Use of enzyme amplification in an ELISA to increase sensitivity of detection of barley yellow dwarf virus in oats and in individual vector aphids

A new technique of alkaline phosphatase amplification in an ELISA (amplified ELISA) was used to increase the sensitivity of detection of barley yellow dwarf virus (BYDV) from oat plant sap and in individual vector aphids. Amplified ELISA differs from conventional direct double antibody sandwich ELISA (DAS-ELISA) in the enzyme substrate reaction. The bound enzyme-labelled antibody catalyzes the conversion of NADP to NAD which is then used in a secondary enzyme-mediated cyclic reaction producing a red-coloured end product. Amplified ELISA was compared with DAS-ELISA for the detection of BYDV and each assay was done with both monoclonal and polyclonal antibody reagents. Both types of antibodies detected BYDV from oat sap and amplified ELISA increased the sensitivity of detection sufficiently to allow a diagnostic test to be completed in less than 2 h using microtitre plates precoated with antibodies. However, in the amplified ELISA using polyclonal antibodies the absorbance values obtained with the healthy oat sap samples were much greater than those obtained in the DAS-ELISA, or with the monoclonal antibodies, and were too large to be acceptable for reliable diagnostic tests. Both monoclonal and polyclonal antibodies were used successfully to detect BYDV in individual virus-carrying Rhopalosiphum padi by amplified ELISA and there was little nonspecific background reaction in the control samples with either of the antibodies.     

Herpes simplex virus detection by ELISA: effect of enzyme amplification, nature of lesion sampled and specimen treatment

The relative sensitivity of two enzyme detection procedures was investigated in a simultaneous "monoclonal" ELISA for herpes simplex virus (HSV). A cyclical enzyme amplified detection system with alkaline phosphatase, rather than horse-radish peroxidase and a conventional chromogenic substrate, gave an increase in absolute sensitivity and a 20 to 30% increase in the detection of HSV in routine isolation-positive genital specimens collected in transport medium. The HSV detection rate, with both procedures, was shown to vary with the site and clinical stage of lesion sampled; it was highest with penile vesicular lesions. Direct extraction of the swab specimen in a small volume of diluent further increased the sensitivity of antigen detection giving positive and negative predictive values of 100 and 96% respectively. The overall sensitivity of HSV detection was equivalent to that obtained by isolation in cell culture. The amplified ELISA offers an alternative, rapid, simple, non-culture technique for routine HSV diagnosis that does not rely upon retention of virus viability.     

Production and use of monoclonal antibodies for the detection of apple chlorotic leaf spot virus

Stable hybridoma cell lines secreting antibodies specific for the apple chlorotic leaf spot virus (CLSV) were produced by fusing spleen cells of a Biozzi mouse immunized with CLSV P863 strain, with the non-secretory P3 X63 Ag8.653 myeloma cell line. Two hybridoma clones producing monoclonal antibodies of the IgG1 subclass were obtained. These monoclonal antibodies were used for virus detection by enzyme-linked immunosorbent assay (ELISA). In contrast to polyclonal antisera to CLSV, which always contain some antibodies to host components, monoclonal antibodies are highly specific for the virus. It was thus possible to develop a detection assay which is more sensitive and specific than the assays using polyclonal antibodies. Using monoclonal antibodies, it was possible to detect less than 0.1 ng/ml of purified virus. In addition, these two monoclonal antibodies recognize 17 strains or isolates maintained in our laboratory and representing most of the known CLSV strains.     

Comparison of polyclonal antiserum versus monoclonal antibodies for the rapid diagnosis of influenza A virus infections by immunofluorescence in clinical specimens.

A pool of monoclonal antibodies was compared with polyclonal antiserum for the rapid detection of influenza A virus in 28 clinical specimens by immunofluorescence. Monoclonal antibodies showed higher sensitivity (69 versus 46%) and accuracy (86 versus 75%) and easier slide interpretation than did polyclonal antiserum. The procedure proved useful for rapid detection of a community outbreak of influenza A virus infection.     

Diagnostic potential of puumala virus nucleocapsid protein expressed in Drosophila melanogaster cells

Puumala virus (PUU) nucleocapsid protein (N) was expressed in insect cells by using the Drosophila Expression System (DES; Invitrogen BV, Groningen, The Netherlands). Stable transfectants were established by hygromycin B selection and showed continuous expression of the recombinant protein (DES-PUU-N) for at least 5 months. The antigenic property of DES-PUU-N was shown to be identical to that of native PUU N when examined with a panel of hantavirus-specific monoclonal antibodies. Enzyme-linked immunosorbent assays (ELISAs) for detection of human immunoglobulin M (IgM) and IgG antibodies were established by using DES-PUU-N as antigen and were compared to assays based on native N. The ELISAs were evaluated for patient diagnosis and seroepidemiological purposes with panels of sera collected from patients with hemorrhagic fever with renal syndrome (HFRS) and from healthy blood donors. Equally high sensitivities and specificities for detection of PUU-specific IgM in acute-phase HFRS patient sera were obtained by the ELISA based on DES-PUU-N and the assay based on the native antigen. For detection of PUU-specific IgG, the ELISA based on monoclonal antibody-captured DES-PUU-N antigen showed optimal sensitivity and specificity.     

Comparison of three monoclonal antibody-based enzyme immunoassays for detection of herpes simplex virus in clinical specimens

Three commercial monoclonal antibody-based enzyme immunoassays (Herpchek, IDEIA HSV and SureCell HSV) for detection of herpes simplex virus antigen were compared with isolation of virus in cell cultures. A total of 51 culture positive and 49 culture negative consecutively collected specimens that had been stored at -70°C for a period of up to ten months were used in the study. Herpchek, IDEIA HSV and Sure-Cell HSV assays gave a sensitivity of 88.2 %, 82.4 % and 47.1 % respectively, and a specificity of 95.9 %, 93.9 % and 83.7 % respectively compared to cell culture. A blocking antibody test showed that two culture negative specimens contained herpes simplex virus-specific antigens. If these two specimens were considered to be true positive, Herpchek, IDEIA HSV and SureCell HSV assays had a sensitivity of 88.7 %, 83.0 % and 47.2 %, and a specificity of 100 %, 97.9 % and 85.1 % respectively. The positive predictive value (using the resolved sample results) for Herpchek, IDEIA HSV and SureCell HSV was 100 %, 97.8 % and 78.1 % respectively, and the negative predictive value 88.7 %, 83.6 % and 58.8 % respectively. These results demonstrated that Herpchek and IDEIA HSV are sensitive and highly specific assays. Results could be obtained in less than five hours after receipt of specimens. SureCell HSV gave results in 15 minutes, but both the sensitivity and specificity were too low for this test to be considered as a substitute for culture.     

Conventional tube cell culture compared with centrifugal inoculation of MRC-5 cells and staining with monoclonal antibodies for detection of herpes simplex virus in clinical specimens.

During a 15-month period, two methods for detection of herpes simplex virus (HSV) in 699 clinical specimens were compared: (i) 24-well-plate centrifugation (24WPC) with MRC-5 cells and staining with type-specific monoclonal antibodies (Syva Co., Palo Alto, Calif.) after incubation for 16 to 18 h and (ii) conventional tube cell culture with primary rabbit kidney and A549 cells. HSV was identified by conventional tube cell culture in 165 (24%) of 699 specimens and by the 24WPC method in 116 (17%) of 699 specimens. One specimen was positive for HSV by the 24WPC method alone, compared with 50 specimens positive only by conventional cell culture (P less than 0.0001). The sensitivity, specificity, and positive and negative predictive values of the 24WPC technique with MRC-5 cells for detection of HSV in clinical specimens were 70, 99.8, 99, and 91%, respectively. Centrifugal inoculation of MRC-5 cells in 24-well plates and staining with monoclonal antibodies after incubation for 16 to 18 h is an insensitive means of detecting HSV in clinical specimens and should not replace conventional tube cell culture with primary rabbit kidney cells.     

Comparison of two commercial enzyme-linked immunosorbent assays for detection of herpes simplex virus antigen

Two commercial enzyme-linked immunosorbent assays (ELISA) for detection of herpes simplex virus (HSV) antigen in direct clinical specimens were compared with cell culture in primary rabbit kidney and MRC-5. With a total of 238 specimens, the sensitivity and specificity of the Dako ELISA method were determined to be 70.1% and 82.4%, respectively, and 60.4% and 88.2%, respectively, for the Ortho Antigen ELISA.     

Biotin-avidin-amplified enzyme immunoassay for detection of herpes simplex virus antigen in clinical specimens

A biotin-avidin-amplified enzyme-linked immunosorbent assay (B-A ELISA) has been developed to detect herpes simplex virus type 1 (HSV-1) and HSV-2 antigens in clinical specimens. The test was designed as a solid-phase, double-antibody, sandwich assay in which plates were coated with a polyclonal rabbit immunoglobulin G anti-HSV reagent, and the sandwich antibody was a biotin-labeled mouse immunoglobulin M monoclonal antibody that reacts with a common antigen associated with HSV-1 and HSV-2. The test can be completed in 4 h if antibody-coated plates are available. The detection limit of the B-A ELISA, determined by titration of virus stocks, was found to be approximately 90 PFU or 6 X 10(3) physical particles of either HSV-1 or HSV-2 per 50 microliter of virus stock. The following results were obtained in a study in which swabs were taken from a variety of lesions and assayed for infectivity in tissue culture and by B-A ELISA. Of 421 suspected HSV lesions tested, 69 were positive by both tests and 159 were negative by both tests. A total of 122 were positive by B-A ELISA but negative for infectivity. Seventy-one were negative by B-A ELISA but contained infectious virus. The HSV specificity of the assay was substantiated by partial blocking of reactivity with rabbit immunoglobulin G anti-HSV and by the absence of reactivity with a nonspecific biotin-labeled mouse immunoglobulin M monoclonal antibody.     

Detection of herpes simplex virus in direct specimens by immunofluorescence assay using a monoclonal antibody.

A monoclonal antibody (MAb), designated CHA 437, was developed against herpes simplex virus (HSV). This MAb (isotype, immunoglobulin G2b K) reacted with HSV type 1 and HSV type 2. It showed no cross-reactivity with varicella-zoster virus, cytomegalovirus, or Epstein-Barr virus. Direct detection of HSV antigen in clinical specimens using indirect immunofluorescence with this MAb was compared with tissue culture isolation. For the 682 specimens tested, the direct specimen test gave a sensitivity of 84.6% and a specificity of 95.7%.     

Alteration in antibody reactivity with foot-and-mouth disease virus (FMDV) 146S antigen before and after binding to a solid phase or complexing with specific antibody

This paper describes the reactions of a number of monoclonal antibodies produced against purified whole virions of foot-and-mouth disease virus in 3 different enzyme immunoassay systems. The first system used whole virus bound non-covalently to microplates; the second used whole virus trapped by a polyclonal antibody which was bound to microplates; and the third allowed the monoclonal antibodies to react with the whole virions in suspension (liquid phase) before trapping by the solid-phase-bound polyclonal antibody. Different reactions with panels of monoclonal antibodies were observed depending on which system was used. Such variations in reactivity give an insight into the alterations in the expression of virus epitopes in the different enzyme immunoassay systems. The reactions of selected monoclonal antibodies were used to illustrate these changes and the results compared to those obtained in similar systems using polyclonal antisera produced against isolated virion polypeptides.     

Rapid diagnosis of herpes simplex virus infections in conventional and shell vial cell cultures using monoclonal antibodies

One hundred specimens for herpes simplex virus (HSV) isolation were tested in parallel by conventional and by centrifugation-enhanced cell culture, followed by identification using monoclonal antibodies to HSV-1 and HSV-2. Sensitivity was comparable by the two methods; conventional culture was only marginally slower and was easier to fit into the routine of a busy laboratory. It is, therefore, advocated for HSV detection in clinical specimens.     

Rapid detection of equine herpesvirus type-1 antigens in nasal swab specimens using an antigen capture enzyme-linked immunosorbent assay.

An antigen capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of equine herpesvirus type-1 (EHV-1) antigens in nasal swab specimens. The test was designed as a solid phase, amplified sandwich assay in which an EHV-1 specific monoclonal antibody was used to capture virus antigen and polyclonal antisera used to detect antigen bound to the test plates. Eight monoclonal antibodies were tested for their ability to capture virus antigen and one was selected for routine use. The sensitivity and specificity of the ELISA was compared with that of virus isolation using swabs from ponies which were experimentally infected with EHV-1. Of 72 nasal swabs collected, 32 were found to be positive by both virus isolation (VI) and ELISA, a further 15 samples were positive by VI alone, but none of the samples were positive by ELISA and negative by VI. This yielded an overall assay sensitivity of 68% and specificity of 100%. The assay proved useful for diagnosis since virus antigen was detected during the first four days post-infection which corresponded to the acute phase of disease when some clinical symptoms were apparent. In addition, the assay could be completed within one day when antibody coated plates were available.     

Evaluation of three immunofluorescence assays for culture confirmation and typing of herpes simplex virus.

Three pairs of monoclonal antibodies, supplied in kits by Electro-Nucleonics, Inc. (ENI), The Syva Co., and Kallestad Laboratories, Inc. (KL), were evaluated for the laboratory confirmation and typing of herpes simplex virus (HSV). Of 108 coded HSV slide preparation, run in parallel with each monoclonal-antibody set, 103 were equivalent by the immunofluorescence assays. Among the five discordant isolates, three (2.8%) did not type with the KL monoclonal antibodies and two (1.9%) false-positive results occurred with the Syva typing system. All of the HSV clinical isolates tested were correctly typed with the ENI indirect immunofluorescence antigen detection system. Typing confirmation of the five discordant HSV isolates was performed by differential sensitivity to 5-bromo-2'-deoxyuridine and endonuclease cleavage analysis of the viral DNA. Use of the Syva and KL direct immunofluorescence antigen detection systems for the identification of HSV isolates is less time-consuming than use of the ENI indirect antigen detection system; however, sensitivity and specificity may be lost.     

Effect of dexamethasone on detection of herpes simplex virus in clinical specimens by conventional cell culture and rapid 24-well plate centrifugation.

During a 4-month period, two methods for rapid detection of herpes simplex virus (HSV) were examined: (i) pretreatment of A549 cells with dexamethasone for conventional tissue culture (277 specimens) and (ii) 24-well plate centrifugation using A549 cells with and without dexamethasone pretreatment and staining with serotype-specific monoclonal antibodies (Syva Co., Palo Alto, Calif.) after incubation for 16 to 18 h (153 specimens). By conventional tube cell culture, both with and without dexamethasone, HSV was identified in 88 of 277 (32%) specimens. Significantly more specimens were positive for HSV at 24 h (46 versus 27 specimens) and at 48 h (a total of 72 versus 59 specimens) (P less than 0.0001) in dexamethasone-treated A549 cells. Of the 153 specimens tested by conventional culture and 24-well plate centrifugation, HSV was detected in 44 (29%) by conventional culture, and by 24-well plate centrifugation with and without dexamethasone, HSV was detected in 32 (21%) and 30 (20%) specimens, respectively. The sensitivity, specificity, and positive and negative predictive values of 24-well plate centrifugation with A549 cells for detection of HSV were 73 (71% without dexamethasone), 100, 100, and 90%, respectively. In conventional tube cell culture, pretreatment of A549 cells with dexamethasone results in more rapid detection of HSV. Centrifugal inoculation of dexamethasone-treated and untreated A549 cells in 24-well plates and staining with monoclonal antibodies after incubation for 16 to 18 h is an insensitive means to detect HSV in clinical specimens and should not replace conventional tube cell culture.     

ELISA for detection of Mycoplasma pneumoniae antigens using monoclonal antibodies

Seven monoclonal antibodies directed against major antigens of Mycoplasma pneumoniae were selected for the development of an antigen detection assay. Three of these were directed to the 170,000-dalton adhesin of M. pneumoniae. The test was an antigen-capture enzyme immunoassay using the different monoclonal antibodies for capture of antigen and a polyclonal rabbit antiserum as detection reagent. With three of the monoclonal antibodies a detection limit of approximately 2 ng M. pneumoniae protein was obtained, as determined by titration of M. pneumoniae organisms in buffer. The detection limit of the assays was only slightly less when the other four monoclonal antibodies were used. In artificially infected nasopharyngeal aspirates the detection limit was approximately 10 times lower. The fact that no significant differences in the detection limit of the assays were recorded using monoclonal antibodies directed against different antigens indicates that these antigens were available for reaction with antibodies irrespective of their location in intact M. pneumoniae cells. In the assay there were no significant cross-reactions with a number of bacterial species potentially colonizing the respiratory tract, except for a protein A-positive strain of Staphylococcus aureus. Our test is equally sensitive to another recently described ELISA using polyclonal antibodies. In comparison with other recommended methods such as immunoblot and culture-amplified antigen detection assays, the ELISA is more rapid and less laborious.     

The use of beta-lactamase in enzyme immunoassays for detection of microbial antigens

The sensitivity and performance characteristics of enzyme immunoassays (EIA) depend to a great extent on the kinetics of the enzyme-substrate system used as indicator. We labeled a variety of polyclonal and monoclonal immunoglobulins with purified beta-lactamase and used them in sensitive EIA systems for the detection of a number of microbial antigens. Polyclonal antibodies to rotavirus, adenovirus, and Haemophilus influenzae type b polyribitol phosphate and monoclonal antibodies to dengue virus were labeled with beta-lactamase and used to provide sensitive direct EIA systems for the detection of the corresponding antigens. In addition, antibodies directed at animal immunoglobulins were labeled with beta-lactamase and used in indirect EIA for the detection of viral antigens with unlabeled anti-viral monoclonal and polyclonal antibodies. Similarly, avidin from Streptomyces was labeled with beta-lactamase and used to detect viral antigens tested for in an avidin-biotin format. Enzyme immunoassay systems with beta-lactamase-labeled antibodies were also used to detect rotaviral and adenoviral antigens in rectal swab specimens from children with acute gastroenteritis. The sensitivity of the beta-lactamase EIA compared favorably with that of analogous EIA systems using alkaline phosphatase or horseradish peroxidase. The results of a beta-lactamase EIA were easily determined by naked eye and a permanent record of the qualitative results obtained by the use of a standard office photocopier, obviating the need for an expensive colorimeter. Enzyme immunoassays using beta-lactamase have potential as practical assay systems for the detection of a wide range of microbial antigens using monoclonal and polyclonal antibodies.     

Quantitation of a cationic antimicrobial granule protein of human polymorphonuclear leukocytes by ELISA

The quantitation of CAP57, a highly hydrophobic, native cationic antigen of human polymorphonuclear leukocytes has been achieved using ELISA. An important feature determining the sensitivity and precision of the ELISA was the reduction of non-specific protein-protein binding, particularly in the inhibition assays, thus eliminating high backgrounds obtained with presently available methodology. Washing of the solid phase-bound antigen and blocking of the non-specific binding sites using a potassium phosphate buffer containing heparin largely contributed to this increased sensitivity. The inhibition assays were conducted using antigen concentrations over the range of 0.9-120 ng. The assay is highly specific and can be performed using monoclonal antibodies and polyclonal antibodies. Non-specific reactions were observed only when high concentrations of antigen (greater than 100 ng) were present in the inhibition mixture. The technique as described is extremely simple, highly reproducible and could be of value in the detection of cationic antimicrobial proteins in the clinical setting in the future.     

Evaluation of biotin-streptavidin enzyme-linked immunosorbent assay for detection of genital herpes simplex virus infection

A biotin-streptavidin enzyme-linked immunosorbent assay (B-SA ELISA) was evaluated for detection of herpes simplex virus (HSV) in clinical specimens which were cervico-vaginal swabs from 205 asymptomatic women and swabs from the genital lesions of 163 suspected patients. All specimens were also subjected to a conventional virus isolation in cell culture. A blocking B-SA ELISA had 100% specificity and 98% sensitivity compared with viral isolation from patients, but had only 40% sensitivity using specimens from asymptomatics. The conventional B-SA ELISA might also be used; it gave results corresponding to B-SA ELISA blocking test except for a single specimen which was considered a false positive.