Comparative sensitivities of solid-phase immune electron microscopy and enzyme-linked immunosorbent assay for serotyping of human rotavirus strains with neutralizing monoclonal antibodies.

Suspensions of 24 rotavirus strains, 6 for each known human rotavirus serotype, were serially diluted and titrated by (i) enzyme-linked immunosorbent assay (ELISA) for rotavirus detection, using monoclonal antibodies (MAbs) specific for group-specific sites of the VP6 inner capsid protein; (ii) ELISA for subgrouping, using MAbs reactive with subgroup-specific determinants of rotavirus VP6; (iii) ELISA for serotyping, using MAbs directed to serotype-specific sites of the VP7 outer capsid glycoprotein; and (iv) solid-phase immune electron microscopy (SPIEM) for serotyping, using VP7-specific MAbs. In addition, in each preparation the proportion of double-shelled rotavirus particles were determined by direct electron microscopy. Results showed that SPIEM was 2- to 16-fold more sensitive than ELISA for serotyping of rotavirus. The titers in VP7-specific tests correlated well with the proportion of double-shelled virus particles in each of the samples. Titers obtained by ELISA for serotyping of suspensions containing 20% or fewer complete particles were up to 4,096-fold lower than those obtained by ELISA for detection. ELISA serotyping titers of samples containing 20 to 80% double-shelled rotavirus particles were up to 128-fold lower than ELISA detection titers, whereas preparations with nearly 100% complete particles had ELISA titers that were less different from each other. ELISA subgrouping titers were four- to eightfold lower than corresponding rotavirus detection titers. It was concluded that, although SPIEM appears to be more sensitive than ELISA, the amount of complete virus particles in the specimens is of critical importance for successful serotyping of human rotavirus strains. Samples rich in single-shelled particles but containing low amounts of VP7 outer capsid glycoprotein might even be strongly reactive in assays for rotavirus detection and subgrouping but virtually unreactive in tests for serotyping.     

Subgrouping of human rotavirus strains by complement fixation,indirect double-antibody sandwich enzyme-linked immunosorbent assay and solid-phase immune electron microscopy

Summary Complement fixation (CF), indirect double-antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) and solid-phase immune electron microscopy (SPIEM) were compared for their ability to subgroup 73 human rotavirus (HRV) strains from infants and young children with gastroenteritis admitted to one or the other of two different hospitals of Northern Italy. By both indirect DAS ELISA and SPIEM all 73 HRV strains were classified into one or the other of two subgroups. By CF only 67 strains could be subgrouped, as six HRV-positive stools showed anticomplementary activity which could not be eliminated. Indirect DAS ELISA required subgroup-specific, unabsorbed antisera from two different animal species. For SPIEM two antisera from a single animal species were needed, but they had to be absorbed with single-shelled bovine rotavirus for HRV subgrouping to be reliable. Indirect DAS ELISA appeared to be the technique most suitable for extensive application in epidemiological studies of HRV infections by different subgroups. However, SPIEM allowed rapid subgrouping of HRV in stool specimens showing anticomplementary activity in the CF test or non-specific reactions in the ELISA test. In one area of Northern Italy the prevalence of subgroup I HRV infections was 7.8 per cent, while in another it reached 68.1 per cent in the same period.With 1 Figure     

Studies on human lacteal rotavirus antibodies by immune electron microscopy

In vitro studies carried out by immune electron microscopy (IEM) indicate that rotavirus aggregation detected in the stools of newborn breast-fed infants with rotavirus infection is antibody-induced. Aggregation of rotavirus particles occurred with the IgA-containing fraction of expressed breast milk (EBM) obtained five days postpartum and with the IgA- and IgG-containing fractions of a pool of EBMs containing samples collected 2-3 days postpartum. Bovine milk fractions also demonstrated this activity in the IgG- and IgA-containing fraction. Studies on unfractionated EBMs from a mother who experienced a rotavirus infection during the 43rd week of lactation showed that following rotavirus infection all three major classes of rotavirus-specific antibodies were present in breast milk, this being confirmed by enzyme immunoassay.     

Solid-phase immune electron microscopy with human immunoglobulin M for serotyping of Norwalk-like viruses.

A solid-phase immune electron microscopy method that uses protein A, goat anti-human immunoglobulin M (IgM), and human serum is described. Evaluation of the method with different immunoglobulin fractions showed that human IgM constituted the major virus capture antibody. The method appeared to distinguish between two Norwalk-like virus serotypes and demonstrated specific IgM responses to these serotypes in infected individuals. Further work is being carried out to define the relationship of these two serotypes to the previously described Norwalk agent (A. Z. Kapikian, R. G. Wyatt, R. Dolin, T. S. Thornhill, A. R. Kalica, and R. M. Chanock, J. Virol. 10:1075-1081, 1972), and four subsequent hospital outbreaks are being studied.     

Enzyme-linked immunosorbent assays adapted for serotyping of human rotavirus strains.

Five enzyme-linked immunosorbent assay systems were adapted for serotyping human rotavirus strains and were compared with a sensitive complement fixation test in terms of specificity and sensitivity. The assays differed mainly with regard to the antibody systems involved in the double sandwich. Serotype differentiation of 34 rotavirus strains was achieved by determining a neutralization endpoint titer, either with a constant antiserum-varying antigen dilution method or vice versa. The procedure which proved to be highly specific and sensitive was one with two type-specific hyperimmune sera (enzyme-linked immunosorbent assay system 5) instead of only one, as in the four other systems.     

Comparison of solid-phase immune electron microscopy by use of protein A with direct electron microscopy and enzyme-linked immunosorbent assay for detection of rotavirus in stool.

A total of 525 stool specimens collected during 1 year were examined for the presence of rotavirus by direct electron microscopy (EM), enzyme-linked immunosorbent assay (ELISA), and a solid-phase immune electron microscope method (SPIEM) utilizing protein A-coated grids for anchoring of specific viral antisera. Rotavirus was seen in 187 specimens; SPIEM detected 183 (97.8%), whereas direct EM and ELISA detected 161 (86%) and 166 (88.7%), respectively. No false-positive reactions were seen by ELISA. The sensitivity of the methods was evaluated by coded investigation of a dilution series of a positive sample, with a negative fecal specimen as diluent. SPIEM was approximately 30 times more sensitive than direct EM and 10 times more sensitive than ELISA. A study was done to compare the elapsed time for recognition of rotavirus by SPIEM and EM in 25 randomly selected positive specimens. All virus-positive specimens were detected within 2 min by SPIEM, whereas up to 9 min was required for direct EM. SPIEM with protein A is a highly sensitive method, useful for rapid detection of viruses in clinical specimens. Due to the direct visualization of virus particles by electron microscopy, there is no requirement for monospecific antisera for the method.     

Comparative evaluation of a commercial enzyme-linked immunoassay and solid-phase immune electron microscopy for rotavirus detection in stool specimens.

Using solid-phase immune electron microscopy (SPIEM) as a reference test, we examined 151 stool specimens from infants and young children with acute gastroenteritis for rotavirus detection by a one-step commercial enzyme-linked immunosorbent assay (ELISA) with labeled monoclonal antibody. Of the 83 samples determined to be positive for rotavirus by SPIEM, 82 were detected as positive by the monoclonal antibody ELISA (sensitivity, 98.7%), while 67 of the 68 specimens determined to be negative by SPIEM were correctly detected as negative by the ELISA (specificity, 98.5%). The diagnostic accuracy of the ELISA kit was 98.6%. Thus, the one-step monoclonal antibody ELISA, which can be completed in less than 90 min, appears to be highly suitable for the rapid and reliable detection of rotavirus in stools.     

Three serotypes of Norwalk-like virus demonstrated by solid-phase immune electron microscopy

Solid phase immune electron microscopy (SPIEM) was used to investigate the serological differences between Norwalk-like virus (NLV) strains from five different outbreaks within the United Kingdom. The existence of two previously demonstrated serotypes, Lewis et al. (Journal of Clinical Microbiology 26:938-942, 1988), was confirmed by the use of whole convalescent sera and purified IgM. A third serotype was found to be the agent of two recent hospital outbreaks and could similarly be typed by use of whole sera or pure IgM. Paired sera were available for two of the three serotypes and demonstrated rising antibody levels. These antibody rises were also specific for the infecting serotype. However, two serum pairs from a later outbreak gave antibody rises to all three serotypes, although much higher counts were produced with the infecting serotype. SPIEM is a useful method for distinguishing NLV serotypes and can also be used to detect specific IgM and to demonstrate seroconversion. Cross-reacting antibodies, possibly anamnestic in origin, can occur after natural infection and could cause confusion in typing virus unless further evidence of the identity of the infecting agent is obtained.     

Sensitive solid-phase immune electron microscopy double-antibody technique with gold-immunoglobulin G complexes for detecting rotavirus in cell culture and feces.

A new solid-phase immune electron microscopy double-antibody colloidal-gold technique (SPIEMDAGT) was developed and compared with direct electron microscopy, direct immune electron microscopy, and enzyme immunoassay for detecting rotavirus. Guinea pig and rabbit antirotavirus antisera were used as capture and detector antibodies, respectively, and goat anti-rabbit immunoglobulin G-gold complexes were employed as a label. Animal rotavirus in cell culture media and human virus in stool specimens were detected by this method. On average, SPIEMDAGT detected 800 times more virus particles than direct electron microscopy and 45 times more particles than direct immune electron microscopy and yielded 20% more positives than enzyme immunoassay. SPIEMDAGT could detect not only viral antigen associated with morphologically recognizable particles but also antigen present when whole virus particles were not visible.     

Rapid high-resolution immune electron microscopy of plant viruses

Two immune electron microscopic methods are described that allow rapid demonstration of (a) specific antibody coating of virus particles or (b) specific clumping and a tenfold increase in the number of particles visible on the grid. The operations take only 20 min and produce high-resolution images not only with purified preparations but also with crude preparations or those containing salts, buffers or sucrose.     

Rapid diagnosis of rotavirus infections: comparison of electron microscopy and immunoelectroosmophoresis for the detection of rotavirus in human infantile gastroenteritis.

Eighty-seven faecal samples from infants and children suffering from acute gastroenteritis were investigated for the presence of rotavirus by immunoelectroosmophoresis (IEOP) and electron microscopy (EM). Sixty-one % of the samples contained rotavirus antigens when examined by IEOP whereas only 50% were diagnosed as positive EM. However, where it was possible to perform EM within the same day that the sample was received it took 24 h to establish the diagnosis by IEOP. The high sensitivity of the IEOP method was achieved by application of antiserum produced in rabbits to rotavirus immunoprecipitates. The specificity and sensitivity of the diagnostic antiserum produced were tested by application of different immunoelectrophoretic methods.     

Serotyping of coxsackieviruses by immune electron microscopy.

Coxsackieviruses B1, B2 and B3 were identified by immune electron microscopy. The results were in good agreement with those of virus neutralization tests in cell cultures.     

Rapid detection of cytomegalovirus using immune scanning electron microscopy

Monoclonal antibodies, specific for human cytomegalovirus, were conjugated to latex microspheres that were already labelled with rabbit anti-mouse immunoglobulin. The beads were then incubated with serum or urine from patients, and then collected on a filter surface, which was analyzed in a scanning electron microscope. Size, immunological specificity, and relative quantity of virus particles were determined within 2 h after serum or urine collection by the visualization of virus particles specifically bound to the latex-bead surface. No such binding of virus particles was detected in the various controls. This method was compared with conventional virus isolation by tissue culture. It enables identification of viruses within a few hours in different body fluids. Even without specific antibodies, the filtration method may permit the rapid detection of particles and the determination of their size in various body fluids.     

Rapid detection of cytomegalovirus using immune scanning electron microscopy

Monoclonal antibodies specific for human cytomegalovirus were conjugated to latex microspheres that were already labelled with rabbit anti-mouse immunoglobulin. The beads were incubated with serum or urine from patients, and then collected on a filter surface, which was analyzed in a scanning electron microscope. Size, immunological specificity, and relative quantity of virus particles were determined within 2 h after serum or urine collection by the visualization of virus particles specifically bound to the latex bead surface. No such binding of virus particles were detected in the various controls. This method was compared with conventional virus isolation by tissue culture. It enables identification of viruses within a few hours in different body fluids. Even without specific antibodies, the filtration method may permit the rapid detection of particles and the determination of their size in various body fluids.     

Hemagglutination by human rotavirus strains

Human rotavirus isolates, KUN , MO, and Wa strains were found to agglutinate erythrocytes of the day-old chicken and adult goose, optimally at pH 6.6. Only those fractions containing double-shelled rotavirus particles isolated by isopycnic centrifugation in cesium chloride had hemagglutinating activity. Trypsin treatment decreased the hemagglutination titer of human rotavirus strains and conversely increased their infectivity. Hemagglutination inhibition tests with antisera against type-specific antigens demonstrable by neutralization showed no type specificity.     

Serotyping of human astrovirus strains by immunogold staining electron microscopy

Human astrovirus strains were propagated in CaCo-2 cell cultures, and virus multiplication was demonstrated by immunosorbent electron microscopy (ISEM). Serotyping of the virus strains was carried out in cell culture fluids or directly in faecal extracts by an indirect immunogold staining (IGS) electron microscopy technique, using specific rabbit antisera against astrovirus types 1–6 as primary antibodies and goat anti-rabbit IgG gold conjugate as secondary antibody. Thirty-seven astrovirus strains were examined, of which 26 grew in the cell cultures in several passages. IGS of the cell-derived viruses showed that 16, 3, 3, and 4 of the strains were types 1, 2, 3, and 4 respectively. Types 5 and 6 were not demonstrated. Eleven strains did not grow in cell cultures, and attempts to serotype these strains by IGS directly in the faecal extracts were unsuccessful, except for one strain which was found to be type 1. The results indicate that IGS may be a specific and suitable method for serotyping astroviruses grown in cell cultures.     

Comparison of four-layer radioimmunoassay and electron microscopy for detection of human rotavirus.

A four-layer radioimmunoassay (RIA) using polystyrene beads as the solid phase, anti-rota guinea pig IgG as primary antibody, anti-rota rabbit IgG as secondary antibody, and 125I-labelled sheep anti-rabbit immunoglobulin as indicator antibody has been developed for the detection of human rotavirus in stool specimens. A comparison was made of the developed RIA, routine electron microscopy, and research electron microscopy of 147 unconcentrated stool specimens from patients with infantile gastroenteritis. In routine electron microscopy 17 (11.6%) false-positive or false-negative results were obtained when compared with research electron microscopy. Each specimen positive in research electron microscopy was positive in RIA, and six additional RIA positives were found from 58 electron microscopy negative specimens. A confirmatory test was necessary to find out marginally positive but nonspecific reactions in RIA. The developed radioimmunoassay is slightly more sensitive than research electron microscopy of unconcentrated stool specimens and considerably more sensitive and more specific than routine electron microscopy.     

Comparison of direct electron microscopy, immune electron microscopy, and rotavirus enzyme-linked immunosorbent assay for detection of gastroenteritis viruses in children.

An approximate 10% suspension in water of the first available stool sample from 411 infants and young children with acute gastroenteritis was examined by electron microscopy (EM) after 2 min of negative staining. This procedure enabled the detection of 88% of the 199 rotavirus infections, all of the 22 adenovirus infections, and 47% of the 15 approximately 27-nm virus infections ultimately detected by a combination of techniques, including immune electron microscopy (IEM) and rotavirus enzyme-linked immunosorbent assay (ELISA). Of the 204 infections detected by direct EM of stools, 76% were detected within 2 min of viewing, and 94% were detected within 6 min of viewing. Type 1 and type 2 rotavirus particles were visualized with approximately equal efficiency, although type 2 rotavirus infections were more common. Rectal swab preparations were clearly inferior to stool preparations for the detection of virus infection by direct EM. IEM examination was required for efficient visualization of viruses in rectal swab specimens. ELISA was the most sensitive method for the detection of rotaviruses; with this method, all infections in which rotavirus particles were visualized by EM or IEM were detected. However, 73% of the 1,834 specimens which were presumptively positive for rotavirus by conventional indirect ELISA proved to be falsely positive on the basis of EM, IEM, blocking ELISA, confirmatory ELISA, or a combination of these methods. False-positive rotavirus ELISA reactions apparently were eliminated when fecal specimens were tested in a modified confirmatory ELISA with a lower dilution of rotavirus-negative (pre-immunization) than rotavirus-positive (post-immunization) capture antibody from the same animal.     

Rapid electron microscopy in oncology.

The case is argued for wider use of electron microscopy as an aid to histological diagnosis in problem cases such as tumours of uncertain histogenesis. In practical terms, electron microscopy can produce results as "immediate" as many special stains in regular use. The cost of providing an effective service can be favourably compared with that of various other diagnostic aids commonly called upon in normal clinical practice. It is suggested that exploitation of this growth area of morphological pathology will enhance the attractions of the discipline of histopathology to talented potential recruits.