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.     

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.     

Development of a biotin-streptavidin-enhanced enzyme-linked immunosorbent assay which uses monoclonal antibodies for detection of group C rotaviruses.

A biotin-streptavidin-enhanced enzyme-linked immunosorbent assay (ELISA) which uses monoclonal antibodies (MAbs) for the detection of group C rotaviruses was developed. An assay in which plates were coated with three pooled MAbs and biotinylated polyclonal immunoglobulin G (IgG) (polyclonal antibody [PAb]) was used as the detector (MAb capture-PAb detector) was found to be the most sensitive and specific of the assays when it was compared with assays in which plates were coated with polyclonal antiserum and detection was done with either biotinylated polyclonal antiserum (PAb capture-PAb detector) or biotinylated pooled MAbs (PAb capture-MAb detector). The MAb capture-PAb detector ELISA detected 83% of samples confirmed to be positive for group C rotaviruses, whereas the PAb capture-PAb detector assay detected 63% of positive samples and the PAb capture-MAb detector assay detected 65% of positive samples. All three procedures detected both of the bovine and the two human group C rotaviruses, but none of the three procedures detected fecal samples containing group A and B rotaviruses or fecal samples negative for group C rotaviruses used in this study. The sensitivity of the MAb capture-PAb detector ELISA was determined by serially diluting fecal group C rotaviruses; antigens were detected in maximal positive dilution ranges of 1:1,000 to 1:3,000 for the samples tested. On the basis of the cell culture immunofluorescence assay infectivity titer of semipurified cell culture-passaged Cowden group C rotavirus, the sensitivity of the MAb capture-PAb detection ELISA for detection of homologous group C rotavirus was 53 fluorescent focus units per ml. Epitope mapping by use of the biotinylated MAbs in competition assay suggested that our MAbs may bind to three different but overlapping epitopes. These results suggest that the MAb capture-PAb detector ELISA can be used to study the epidemiology of group C rotaviruses in humans and animals.     

An ELISA method for detecting antibodies to the T cell antigen receptor

An ELISA method for the detection of monoclonal antibodies (MAb) to the T3-T cell antigen receptor (TCR) complex was devised. The T3-TCR complex was solubilised using digitonin and a rat anti-T3 MAb (Campath 3) was used to bind it to an ELISA plate. Normal rat serum was used to block cross-reactivity between the rat MAb and peroxidase-conjugated rabbit anti-mouse immunoglobulins. The assay was tested on four T cell tumour lines and successfully detected MAbs to TCR beta chain variable regions, as well as the anti-T3 MAb UCHT1. Other anti-T3 MAbs were not detected because Campath 3 blocked their binding. None of a panel of MAbs reacting with other T cell surface antigens reacted in the assay.     

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.     

Multiple copies of identical epitopes on the adenovirus hexon

A double monoclonal antibody (MAb) sandwich enzyme-linked immunosorbent assay (double MAb ELISA), which uses the same MAb as solid-phase immunosorbent (capture MAb) and as detector MAb (peroxidase-labeled), was developed to quantify the specific epitopes of adenovirus hexon. Four MAbs directed against crystallized adenovirus type 1 (Ad h 1) hexon were tested by this assay with homologous and different heterologous hexons. The lowest reacting concn with the homologous and heterologous hexon types both in direct and double MAb ELISA was determined and compared. At least two copies of four different epitopes were identified by the MAbs. Evidence is presented that more than one copy of identical or closely related epitopes exist on the homologous as well as on the heterologous hexon molecules. However, their presence could be detected only in higher concn of hexon preparations of subgenera A, B and D.     

Comparisons of rotavirus VP7-typing monoclonal antibodies by competition binding assay.

Three sets of neutralizing monoclonal antibodies (MAbs) used to type the outer capsid protein VP7 of four group A rotavirus serotypes (1 through 4) were compared in competition immunoassays. Reciprocal competition was observed for each of the VP7 type 2-, 3-, and 4-specific MAbs. The VP7 type 1 MAbs exhibited variable competition patterns with other VP7 type 1 MAbs. MAb RV4:3, which has been used to recognize antigenic variants within VP7 type 1 strains, showed reciprocal competition with the four VP7 type 3 MAbs (RV3:1, YO-1E2, 4F8, and 159) using a VP7 type 3 virus (SA11) as antigen. MAb 2C9, also prepared against VP7 type 1, reacted with VP7 type 3 strains and competed with a VP7 type 3 MAb, 159, using RRV as antigen. Use of the different sets of VP7 type-specific MAbs in the enzyme-linked immunosorbent assay permitted the recognition of six antigenic variants within VP7 types 1, 2, and 3 among specimens whose VP7 type could not be determined previously with only one set of typing MAbs. These results demonstrate differences of typing ability among these VP7-specific MAbs and emphasize the need to improve the sensitivity of typing systems by incorporating panels of MAbs reacting with several neutralizing epitopes.     

Production of monoclonal antibodies against recombinant human interferon-gamma: screening of hybridomas without purified antigen

A panel of mouse monoclonal antibodies (MAbs) against the recombinant human gamma interferon (HuIFN-gamma) has been produced for the study of the structure-function relationships of this important lymphokine. Enzyme linked immunosorbent assay (ELISA) is the current method of choice to screen hybridomas for specific MAb production. The purity of the antigen used for screening dictates the specificity of the ELISA. As often is the case in many systems, adequately purified biologically active HuIFN-gamma was not readily available for this purpose. A sandwich ELISA which allowed the use of unpurified HuIFN-gamma for hybridoma screening was developed. A rabbit antiserum against the denatured HuIFN-gamma purified by SDS-PAGE was prepared and the nonspecific binding activity was removed by adsorption to control cell proteins immobilized on Sepharose. The adsorbed immunoglobulin fraction was bound to the ELISA plate: (i) to trap HuIFN-gamma specifically from the whole cell lysate, thus providing specificity for MAb detection, and (ii) to avoid direct adsorption of the HuIFN-gamma to the ELISA plate because others have found that this prevented detection of neutralizing MAb. The sandwich ELISA detected both neutralizing and non-neutralizing MAbs with relatively low false positive reactions. This approach to the development of an ELISA method to screen hybridomas without purified antigen should be applicable to the production of MAbs to other proteins.     

Characterization and potential diagnostic application of monoclonal antibodies specific to rabies virus

Objective

Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem. It is necessary to develop standard rabies virus diagnostic tools, especially for diagnosing the strains prevalent in China.

Methods

Monoclonal antibodies (MAbs) specific to rabies virus were produced and characterized by enzyme linked immunosorbent assay (ELISA), isotyping, affinity assay, immunofluorescence assay (IFA), and immunocytochemistry. The MAb, whose affinity was higher for antigen, was used to establish an antigen capture-ELISA (AC-ELISA) detection system and test the efficiency by using clinical samples.

Results

The heavy chain subclasses of two MAbs were all determined to be IgG2a. The 3C7 MAb showed stronger reactivity with rabies virus protein than the 2C5 MAb in an ELISA analysis, whereas the 3C7 MAb showed the highest affinity for antigen. IFA and immunocytochemistry results also indicated that the two MAbs could recognize rabies virus protein in its native form in cell samples. Data obtained using clinical samples showed that rabies virus could be detected by AC-ELISA detection system using the 3C7 MAb.

Conclusion

It was potentially useful for the further development of highly sensitive, easily handled, and relatively rapid detection kits/tools for rabies surveillance in those areas where rabies is endemic, especially in China.     

Monoclonal antibodies to surface antigens of Mycobacterium tuberculosis and their use in a modified enzyme-linked immunosorbent spot assay for detection of mycobacteria.

Three monoclonal antibodies (MAbs) were generated from splenocytes of a BALB/c mouse immunized with heat-killed Mycobacterium tuberculosis. All three MAbs bound to surface epitopes of M. tuberculosis as shown by whole-cell enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence, and immunoelectron microscopy. One immunoglobulin M (IgM) MAb bound to lipoarabinomannan, the second IgM MAb bound to mycolyl-arabinogalactan-peptidoglycan complex, and the third MAb, an IgG3, bound to a surface epitope of an uncertain nature. The MAbs demonstrated different cross-reactivity patterns with other mycobacteria. Two of the MAbs were used to develop a modified ELISA spot assay for the detection of mycobacteria.     

Characterization of monoclonal antibodies to human group B rotavirus and their use in an antigen detection enzyme-linked immunosorbent assay

Three monoclonal antibodies (MAbs)--B5C9, B5E4, and B10G10--to human group B rotavirus, an agent implicated in epidemic outbreaks of diarrhea in the People's Republic of China, primarily in adults, were prepared. MAb reactivity was decreased when virus preparations were treated with EDTA, suggesting reactivity with the outer-capsid protein(s). Competition experiments suggested that these MAbs recognize overlapping epitopes within a single antigenic site. A simple antigen detection enzyme-linked immunosorbent assay (ELISA) specific for the human group B rotavirus was established by using these MAbs as capture antibodies. Fifteen clinical samples obtained from three epidemic areas in the People's Republic of China and previously shown by Chinese scientists to contain group B virus were all positive in the MAb capture antigen detection ELISA, whereas none of the 57 samples lacking the group B virus reacted in the test. The results suggest that this MAb capture antigen detection ELISA will be useful to identify outbreaks caused by the human group B rotavirus and to monitor possible spread of the virus.     

Antigen-capture ELISA for the detection of Rift Valley fever virus nucleoprotein using new monoclonal antibodies

Monoclonal antibodies (MAbs) raised against the nucleoprotein (NP) of Rift Valley fever virus (RVFV) were developed, and an antigen-capture enzyme-linked immunosorbent assay (Ag-capture ELISA) system was developed for the detection of RVFV NP. The assay detected RVFV antigen from culture supernatants containing as little as 7.8-31.3 pfu per 100 μl. Reactivity with various truncated NPs indicated that MAb C10-54 bound only to the full-length NP, probably due to recognition of a conformational epitope, whereas MAbs G2-36 and D5-59 bound to a linear epitope ranging from amino acid residues 195-201 in the C-terminal region. Based on the alignments of the amino acid sequence of RVFV NP, the epitope regions of MAbs G2-36 and D5-59 were completely conserved among all RVFV strains. These results suggest that the MAbs are applicable to the Ag-capture ELISA for the diagnosis of RVFV infections.     

Antigens in electron-dense granules from Entamoeba histolytica as possible markers for pathogenicity.

In vitro interaction of Entamoeba histolytica with collagen induces intracellular formation and release of electron-dense granules (EDG) and stimulation of collagenolytic activity. Purified EDG contain 1.66 U of collagenase per mg of protein. Thus, EDG may participate in tissue destruction during invasive amebiasis. Monoclonal antibodies (MAbs) L1.1 and L7.1 reacted specifically with EDG in enzyme-linked immunosorbent assay (ELISA) and immunofluorescence and immunoelectron microscopy. MAb L7.1 immunoprecipitated three polypeptides with molecular weights of 95,000, 68,000, and 28,000 from lysates of biosynthetically labeled E. histolytica. Both MAbs recognized the pathogenic E. histolytica axenic strains HM1:IMSS, HM38:IMSS, and HK-9 but failed to react in ELISA with Entamoeba moshkovskii, Entamoeba invadens, and E. histolytica-like Laredo. In addition, MAb L7.1 reacted with one E. histolytica isolate from a symptomatic patient but did not react with four of five isolates from asymptomatic patients. EDG antigens were detected by a MAb L7.1-based ELISA in E. histolytica-containing fecal samples from symptomatic, but not asymptomatic, individuals. These results suggest that the EDG antigen detected with MAb L7.1 may be differentially expressed in pathogenic and nonpathogenic E. histolytica.     

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     

Monoclonal antibody-based capture enzyme immunoassays for specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to respiratory syncytial virus

Monoclonal antibodies (MAbs) to the fusion protein (F), attachment protein (G), and nucleoprotein (N) of respiratory syncytial (RS) virus were evaluated for use as detector antibodies in immunoglobulin G (IgG), IgA, and IgM capture enzyme immunoassays. MAb assays were tested against assays using polyclonal antibodies (PAbs) with serum specimens from patients with and without evidence of recent RS virus infection. Assays developed with N MAbs were comparable to or better than PAb assays for detecting specific IgG and IgM antibodies but were somewhat less sensitive for IgA. F MAb assays were less sensitive for IgG and IgM antibodies but identified specific IgA in some specimens negative by N MAb assay. G MAb assays were insensitive for IgG and IgM antibodies but did detect about 50% of the IgA antibodies identified by the PAb assay. The basis for the low sensitivity of the G MAb assays is unclear, since many of these specimens were positive for IgG antibodies to G by Western immunoblot. The sensitivity of MAb assays varied with patient age: N MAb assays detected specific antibody responses to RS virus in all immunoglobulin classes in both adults and infants less than 1 year of age, F MAb assays detected specific IgG responses in adults and IgA responses in both adults and infants, and G MAb assays only detected IgA responses in adults. A mixture of N and F MAbs was complementary overall, identifying 54 of 55 (IgG), 51 of 52 (IgA), and 16 of 17 (IgM) serum specimens positive by PAb assay. These MAb assays were also specific with specimens tested from persons without a history of recent RS virus infection. The availability of these MAb-based assays offers other laboratories the opportunity to have long-term, standardized reagents and tests for serological diagnosis of RS virus infection.     

Monoclonal antibody characterization of Jamestown Canyon (California serogroup) virus topotypes isolated in Canada.

Jamestown Canyon (JC) virus of the California (CAL) serogroup has been isolated in 12 American states and 6 Canadian provinces. A study was undertaken to produce monoclonal antibodies (MAbs) to JC virus and to use these MAbs to assay for possible heterogeneity among naturally occurring JC topotypes in Canada. MAbs were produced to the prototype strain of JC virus using BALB/c mice. Twenty-seven secreting MAbs were obtained and three of these MAbs were propagated and studied. All three MAbs, M1 (IgG1), M2 (IgG2b), and M3 (IgG2a), were reactive by immunofluorescent antibody assay against JC-infected vero cells and by ELISA against JC antigen. MAb M2 reacted with all members of the Melao complex, MAb M1 reacted only with Keystone virus, while MAb M3 exhibited no reactivity with other CAL serogroup viruses. Only MAb M3 possessed neutralization and hemagglutination inhibition activities against JC virus. The MAbs were also tested by ELISA and for neutralizing activity against 13 JC topotypes isolated in 5 provinces from Newfoundland to Saskatchewan. ELISA confirmed closer identity of the Canadian topotypes to JC as opposed to the closely related South River virus. The MAbs verified all Canadian topotypes to be JC virus but revealed different patterns of reactivity between these topotypes and prototype JC virus.     

Evaluation of combined commercial enzyme-linked immunosorbent assay for detection of rota and adenoviruses for automation

219 stools were examined by direct electron microscopy (EM), culture and ‘combined’ commercial enzyme-linked immunosorbent assay kits (CELISA). The specificity of the combined ELISA for rotavirus was 100% as compared with EM, and 100% for adenovirus when both culture in addition to EM were carried out. ELISA appeared to be more sensitive than EM for both viruses. There was no cross-reaction between the 2 ‘combined’ antisera. This technique may be useful for automation of viral diagnosis with ELISA using a ‘panel’ of selected viruses for a variety of specimens.     

A simple approach to improving sensitivity in a one-step monoclonal antibody-based ELISA for human IIbIIIa using multiple conjugates.

This paper describes an approach which can be used to increase both the speed and the sensitivity of a monoclonal antibody (MAb) based ELISA for human IIbIIIa by using a mixture of enzyme labeled MAbs (conjugates) and a one step incubation format (where analyte and conjugates were incubated simultaneously for 2 h in the MAb coated well). A simple competitive blocking method is described for mapping the relative epitopes of five monoclonal antibodies (MAbs) with minimum preparation of enzyme conjugate. This method permits the selection of three MAbs which recognize distinctly different epitopes, and are suitable for use in a one step ELISA format. Compared to the regular two step ELISA using two MAbs, this simple modification improves the assay sensitivity by three-fold and decreases the incubation time by 75%, while maintaining a high level of precision (2-15%). The assay is very specific and can accurately measure both recombinant and native IIbIIIa.