Validation of a monoclonal antibody-based capture enzyme-linked immunosorbent assay for detection of Campylobacter fetus

A monoclonal antibody (MAb)-based antigen capture enzyme-linked immunosorbent assay (ELISA) was compared with the routine culture methodology for the detection of Campylobacter fetus subspecies from bovine and ovine field samples inoculated into Clark's transport enrichment medium (TEM). The work was a collaboration between two different diagnostic laboratories, one in Canada and the other in England. In both labs, TEM samples were incubated for 4 days at 35 degrees C and then tested by culture and ELISA. The ELISA consisted of initial screening with MAb M1825 against C. fetus subspecies core lipopolysaccharide (LPS). All samples positive on ELISA screening were then retested by ELISA with MAb M1825 and MAbs M1177, M1183, and M1194, which recognize serotype A- and/or serotype B-specific C. fetus subspecies LPS epitopes. The Canadian samples consisted of 1,060 preputial washings from 529 bulls, of which 18 were positive by both culture and ELISA and 1,042 were negative by both methods. The English samples consisted of 321 tissue specimens, mostly stomach contents and placentas, from 190 aborted ovine and bovine fetuses. A total of 262 samples were negative by culture and ELISA, 52 samples were positive by culture and ELISA, and 7 samples were culture negative but ELISA positive. The results for all 70 culture-positive isolates were confirmed by conventional biochemical methods as C. fetus subsp. fetus, with 39 presumptively identified by the ELISA as serotype A and 30 presumptively identified as serotype B and with one sample containing isolates presumptively identified as serotype A and serotype B. A receiver operating characteristic analysis of the combined ELISA data from both countries resulted in an area under the curve of 0.997, with a sensitivity of 100% and a specificity of 99.5% relative to the results of culture. The data confirm that this ELISA method can be used as an excellent test for the screening of field samples in TEM for the presence of C. fetus subspecies.     

Development of an enzyme-linked immunosorbent assay for serotyping ureaplasma urealyticum strains using monoclonal antibodies

Ureaplasma urealyticum comprises 14 serotypes. The existing serotyping methods all use polyclonal antibodies. These methods are time-consuming and labor-intensive, and they cannot always be performed on primary isolates; in addition, the results are difficult to interpret. We developed a new enzyme-linked immunosorbent assay (ELISA) method using serotype-specific monoclonal antibodies (MAbs) to enable the serotyping of U. urealyticum isolates from primary broth cultures. Each of the 14 serotype reference strains was tested against 14 selected MAbs. Homologous reactions were very strong, while heterologous reactions were negligible. Three cross-reactions were observed: MAb 5 cross-reacted with serotype 2, MAb 14 cross-reacted with serotype 3, and MAb 8 cross-reacted with serotype 13. Despite the cross-reactions observed, all the serotype reference strains of U. urealyticum could be identified and differentiated using a combination of MAbs. Reproducibility was analyzed with a fractionated antigenic preparation and with several freshly prepared antigens of the same strain. No significant interrun variation was found with the fractionated antigen, but significant variations in optical density (OD) values were found when freshly prepared antigens were tested. However, the variation in OD values did not influence the overall interpretation of the ELISA: reactions with homologous MAbs were always prominent compared to those of the negative controls. This newly developed ELISA using MAbs seems promising for serotyping of U. urealyticum clinical isolates.     

Validation of a Monoclonal Antibody-Based Capture Enzyme-Linked Immunosorbent Assay for Detection of Campylobacter fetus

A monoclonal antibody (MAb)-based antigen capture enzyme-linked immunosorbent assay (ELISA) was compared with the routine culture methodology for the detection of Campylobacter fetus subspecies from bovine and ovine field samples inoculated into Clark's transport enrichment medium (TEM). The work was a collaboration between two different diagnostic laboratories, one in Canada and the other in England. In both labs, TEM samples were incubated for 4 days at 35°C and then tested by culture and ELISA. The ELISA consisted of initial screening with MAb M1825 against C. fetus subspecies core lipopolysaccharide (LPS). All samples positive on ELISA screening were then retested by ELISA with MAb M1825 and MAbs M1177, M1183, and M1194, which recognize serotype A- and/or serotype B-specific C. fetus subspecies LPS epitopes. The Canadian samples consisted of 1,060 preputial washings from 529 bulls, of which 18 were positive by both culture and ELISA and 1,042 were negative by both methods. The English samples consisted of 321 tissue specimens, mostly stomach contents and placentas, from 190 aborted ovine and bovine fetuses. A total of 262 samples were negative by culture and ELISA, 52 samples were positive by culture and ELISA, and 7 samples were culture negative but ELISA positive. The results for all 70 culture-positive isolates were confirmed by conventional biochemical methods as C. fetus subsp. fetus, with 39 presumptively identified by the ELISA as serotype A and 30 presumptively identified as serotype B and with one sample containing isolates presumptively identified as serotype A and serotype B. A receiver operating characteristic analysis of the combined ELISA data from both countries resulted in an area under the curve of 0.997, with a sensitivity of 100% and a specificity of 99.5% relative to the results of culture. The data confirm that this ELISA method can be used as an excellent test for the screening of field samples in TEM for the presence of C. fetus subspecies.     

Development of an Enzyme-Linked Immunosorbent Assay for Serotyping Ureaplasma urealyticum Strains Using Monoclonal Antibodies

Ureaplasma urealyticum comprises 14 serotypes. The existing serotyping methods all use polyclonal antibodies. These methods are time-consuming and labor-intensive, and they cannot always be performed on primary isolates; in addition, the results are difficult to interpret. We developed a new enzyme-linked immunosorbent assay (ELISA) method using serotype-specific monoclonal antibodies (MAbs) to enable the serotyping of U. urealyticum isolates from primary broth cultures. Each of the 14 serotype reference strains was tested against 14 selected MAbs. Homologous reactions were very strong, while heterologous reactions were negligible. Three cross-reactions were observed: MAb 5 cross-reacted with serotype 2, MAb 14 cross-reacted with serotype 3, and MAb 8 cross-reacted with serotype 13. Despite the cross-reactions observed, all the serotype reference strains of U. urealyticum could be identified and differentiated using a combination of MAbs. Reproducibility was analyzed with a fractionated antigenic preparation and with several freshly prepared antigens of the same strain. No significant interrun variation was found with the fractionated antigen, but significant variations in optical density (OD) values were found when freshly prepared antigens were tested. However, the variation in OD values did not influence the overall interpretation of the ELISA: reactions with homologous MAbs were always prominent compared to those of the negative controls. This newly developed ELISA using MAbs seems promising for serotyping of U. urealyticum clinical isolates.     

Generation and characterization of monoclonal antibodies against Staphylococcus aureus thermonuclease

Monoclonal antibodies (MAbs) against Staphylococcus aureus thermonuclease (TN) were raised by immunizing BALB/c mice with a commercial TN preparation. Six monoclones were generated producing MAbs specific for S. aureus TN as tested in Western blots and ELISA. They all combined with a 17 kD and a 21 kD protein, respectively, both of which showed DNase activity. All MAbs were of IgG1 isotype with kappa light chain. Competition ELISA showed that five of the MAbs recognized a total of three different binding sites of TN, designated I, II and III, respectively. Only the anti-site II MAbs inhibited the DNase activity. A MAb-based sandwich ELISA showed a lower detection limit for TN of approximately 0.5 ng/ml protein. Only S. aureus strains (culture supernatants) showed positive ELISA (31 positive/31 tested), although other tested gram positive cocci produced thermostable nucleases. The MAbs have potentials as reagents for rapid and specific detection of S. aureus.     

Production and characterization of monoclonal antibodies specific for lipooligosaccharide of Serpulina hyodysenteriae.

Serpulina (Treponema) hyodysenteriae is the causative agent of swine dysentery, a contagious mucohemorrhagic disease of the colon. Diagnosis of swine dysentery is extremely difficult because of the presence of cross-reactive antibodies to the proteins of S. hyodysenteriae and Serpulina innocens, a nonpathogenic inhabitant of the porcine large intestine. Therefore, monoclonal antibodies (MAbs) against the serotype-specific lipooligosaccharide (LOS) antigens of S. hyodysenteriae were produced to rapidly differentiate S. hyodysenteriae from S. innocens. Whole-cell preparations of S. hyodysenteriae serotypes 1 through 7 were used as antigens. MAbs were characterized by an indirect enzyme-linked immunosorbent assay with whole-cell or LOS antigen and by Western blot (immunoblot) analysis with whole-cell lysates as antigen. A total of 12 LOS-specific MAbs which could identify and differentiate the seven original serotypes of S. hyodysenteriae were produced. The MAb serospecificities are as follows: MAb 9G8, serotype 1; MAb 31D9, serotype 2; MAb 7D3, serotypes 2 and 7; MAb 24B7, serotype 3; MAb 13C2, serotype 4; MAb 18E9, serotype 4; MAb 2B7, serotype 6; MAb 1D2, serotypes 2, 5, and 7; MAb 9C5, serotypes 2, 5, and 7; MAb 11C9, serotype 7; MAb 11E10, serotype 7; and MAb 6G11, serotype 7.     

Inhibitory activity on bacterial motility and in vivo protective activity of human monoclonal antibodies against flagella of Pseudomonas aeruginosa.

Three stable hybridoma cell lines, IN-2A8, IN-5D6, and ZI-3A8, that secrete human monoclonal antibodies (MAbs) specific for b-type flagella of Pseudomonas aeruginosa were established by fusing peripheral blood lymphocytes from healthy volunteers with murine myeloma P3X63-Ag8.653 cells. The immunoglobulin M MAbs reacted specifically with flagellin (Mr, 52,000) by Western blotting (immunoblotting) analysis and bound specifically to clinical isolates belonging to Homma serotypes A, B, H, I, and M at frequencies of 58, 50, 46, 30, and 35%, respectively, but did not bind to any serotype E or G isolates. Overall, the MAbs bound to 31% of the clinical isolates. MAb IN-2A8 strongly protected burned mice challenged with P. aeruginosa bearing b-type flagella from death following parenteral administration of 0.1 microgram per mouse. This MAb also inhibited P. aeruginosa colony spreading in soft agar at a concentration of more than 1 microgram/ml but only slightly enhanced opsonophagocytosis by human polymorphonuclear leukocytes. A line of evidence suggests that the potent in vivo activity of MAb IN-2A8 in the burned-mouse model is likely to be caused by its inhibition of bacterial motility after binding to flagella.     

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.     

Rapid identification of Staphylococcus aureus by using lysostaphin sensitivity.

Lysostaphin sensitivity was evaluated as a rapid screening test to differentiate Staphylococcus aureus from other species of staphylococci and micrococci. A total of 168 strains of staphylococci, 108 of which were S. aureus, were cultured overnight in brain infusion broth. Gram stains were peformed before and after a 1:10 dilution of the culture was exposed to 2 micrograms of lysostaphin per ml at 37 degrees C for 30 min. A reduction of 90% or greater in the number of organisms seen on comparison of the pre- and posttreatment Gram stains was considered a "positive" test result and was found in 106 of 108 S. aureus strains; 60 of 60 non-S. aureus staphylococci had a negative test result, showing no difference between the pre- and posttreatment Gram stains. Identical results were obtained using commerical blood culture media in place of brain heart infusion broth. Also studied prospectively were 100 blood or broth cultures which the clinical microbiology laboratory identified as containing gram-positive cocci suggestive of staphylococci. All 33 cultures later found to contain S. aureus gave positive test results; 67 of 67 non-S. aureus staphylococci, micrococci, and steptococci were negative.     

Detection of Salmonella spp. in clinical specimens by capture enzyme-linked immunosorbent assay.

A capture enzyme-linked immunosorbent assay (ELISA; Kirkegaard and Perry Laboratories, Gaithersburg, Md.) was used to detect Salmonella spp. in clinical and artificially inoculated specimens. In patients with bacteremia caused by Salmonella spp., 48% (12 of 25) and 82% (13 of 16) of serum and urine specimens, respectively, were positive for Salmonella spp., as determined by ELISA. All serum and urine specimens collected from healthy individuals (25 specimens) or patients whose blood cultures grew gram-negative bacteria other than Salmonella spp. (18 specimens) were negative for Salmonella spp., as determined by ELISA. For blood culture bottles in which Salmonella spp. (16 specimens) was grown the ELISA was positive (100%), while it was negative for all the 65 blood culture bottles in which gram-negative bacteria other than Salmonella spp. (42 specimens) or gram-positive bacteria (23 specimens) were grown. All samples of urine (16 specimens), stool (8 specimens), serum (16 specimens), culture media (12 specimens), and blood culture bottles (reported sterile after 2 weeks of incubation; 16 specimens) that were artificially inoculated with 10(3) to 10(7) CFU of four species of Salmonella per ml were positive by ELISA. Similar specimens inoculated with or containing various species other than Salmonella were negative by this test. Thus, ELISA offers a promising opportunity for the rapid detection of Salmonella spp. in clinical microbiology laboratories.     

Protection against infection with Neisseria meningitidis group B serotype 2b by passive immunization with serotype-specific monoclonal antibody

Hybridomas derived from mice immunized with Neisseria meningitidis serogroup B serotype 2b (B,2b) outer membrane preparations produced monoclonal antibodies (MAbs) specific for major outer membrane proteins of classes 1, 2, and 5. The MAbs were examined by enzyme-linked immunosorbent assay against a selected panel of seven strains of N. meningitidis (B,2b) of different sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns, a serotype 2a, and a nontypable strain. The five MAbs selected were all bactericidal and of different immunoglobulin subclasses. None of the MAbs reacted with other bacterial strains in a dot-enzyme immunoassay. The corresponding antigenic determinant for each MAb was localized on a specific outer membrane protein by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of major outer membrane proteins. MAbs M5-11 and M5-30 bound to the class 2 protein and were serotype 2b specific. MAb M2-20 bound to the class 1 protein, and MAbs M5-16 and M5-19 bound to the class 5 protein. A mouse model of infection was established whereby a local infection progressed to lethal bacteremia over 3 days, and 50% of the animals were killed with an intraperitoneal injection of 10 meningococci plus 4% mucin and 1.6% hemoglobin. The ability of the MAbs to provide passive protection against experimental infection with N. meningitidis (B,2b) was examined. Both serotype-specific MAbs M5-11 and M5-30 were highly protective even though they were of different immunoglobulin subclasses. The class 5-specific MAb offered no protection, while the class 1-specific MAb gave limited protection. It may therefore be possible to provide protection against serotype 2b infection by using as vaccine the class 2 serotype-specific surface-exposed outer membrane protein epitopes defined by MAb M5-11 or M5-30.     

Utility of Hybrisep in the diagnosis of blood stream infection

Hybrisep is an in situ hybridization (ISH) method to detect phagocytosed bacteria in peripheral blood neutrophils and macrophages. We report 10 actual clinical cases tested using Hybrisep with new DNA probes, and the data were compared to the actual blood culture results. A normal Hybrisep strategy employs 5 DNA probes to detect the following bacterial DNA: "SA" probe for S. aureus, "SE" for S. epidermidis, "PA" for P. aeruginosa, "EF" for E. faecalis, and "EK" for E. coli, E. cloacae, and K. pneumoniae. Six newly designed DNA probes were used in this study: "GB" probe for 49 common bacteria, "SP" for S. pneumoniae, "BF" for B. fragilis, "HI" for H. influenzae, "GC" for Candida species, and "CA" for C. albicans. Three cases were positive on ISH, but all their blood cultures were negative. One case showed a positive blood culture, but was negative on ISH. In another 6 cases, both were negative. We postulated that empirical therapy of antibiotics resulted in only positive ISH. Cases only showing a positive outcome on blood culture might be due to a diminished phagocytic function during patients' severe disease conditions. In conclusion, ISH with Hybrisep has clinical advantages such as being able to defect causative pathogens even after the use of antibiotics, and facilitates more rapid identification than routinely performed bacterial cultures only.     

Isolation and characterization of monoclonal antibodies specific for antigen P1, a major surface protein of mutans streptococci.

A panel of 15 murine monoclonal antibodies (MAbs; 14 immunoglobulin G1, 1 immunoglobulin G2a) directed against antigen P1, a major surface protein of mutans streptococci, was prepared. All of these MAbs reacted by the enzyme-linked immunosorbent assay with solubilized wall material from Streptococcus mutans Ingbritt 175 (a serotype c strain which retains significant amounts of P1 in its cell wall), culture supernatant fluid from Ingbritt 162 (a strain which excretes large amounts of P1 into the culture medium), and purified P1. By Western immunoblotting, these MAbs were observed to react with a high-molecular-weight polypeptide which comigrated with antigen P1. None of these MAbs cross-reacted with human heart tissue or with various eucaryotic proteins. When whole cells of various strains of mutans streptococci were screened against the panel of MAbs, the strongest reactivities were noted with strains of serotype c and e S. mutans, while a serotype f strain of S. mutans, along with S. sobrinus and S. cricetus strains, reacted somewhat more weakly. S. rattus strains were completely negative. Results obtained with bacterial culture supernatants were qualitatively similar. The surface localization of antigen P1 was confirmed by electron microscopy with an indirect immunogold technique. In sectioned S. mutans cells, labeling appeared to be associated with a fibrillar "fuzzy coat" layer, which was far more prominent on cells of Ingbritt 175 than on those of Ingbritt 162.     

Production and characterization of monoclonal antibodies specific for Cryptococcus neoformans capsular polysaccharide.

Cryptococcus neoformans is surrounded by a capsular polysaccharide. There are at least four known serotypes of the polysaccharide. The objective of this study was to produce monoclonal antibodies (MAbs) that could be used to study the distribution of epitopes among the serotypes of C. neoformans. BALB/c mice were immunized with cryptococcal polysaccharides of serotype A or D that were coupled to sheep erythrocytes. Splenocytes were isolated, and hybridomas secreting MAbs specific for cryptococcal polysaccharides were isolated. Two hybridomas, designated MAbs 439 and 1255, were produced from mice immunized with serotype A polysaccharide. One hybridoma, designated MAb 302, was produced from mice immunized with serotype D polysaccharide. All three antibodies were of the immunoglobulin G1 isotype. MAb 302 showed a specificity for serotypes A and D in Ouchterlony diffusion, agglutination, and opsonophagocytosis assays. MAb 1255 was reactive with polysaccharides and cells of serotypes A, B, and D. MAb 439 was reactive with polysaccharides and cells of serotypes A, B, C, and D. The reactivity of these MAbs closely matched the distribution of epitopes among cryptococcal polysaccharides predicted in previous studies of polyclonal antibodies reactive with cryptococcal polysaccharides. The ability to produce a MAb against an epitope shared by all four serotypes may have value for the detection of cryptococcal antigens in body fluids.     

Evidence of rotavirus AU32 like G9 strains from nontypeable fecal specimens of Indian children hospitalized during 1993-1994

Serotyping of 432 rotavirus positive fecal specimens collected from hospitalized children during 1990-1997 was carried out at National Institute of Virology (NIV), Pune, India, using monoclonal antibodies (MAbs) directed against VP7 determinant of serotypes G1-G4, G6, G8, and G10. However, significant number of specimens, that is, 47.92% remained nontypeable. The aim of the present study was to culture adapt the nontypeable specimens and to characterize them further. Since the fecal specimens were not tested by MAb to G9 serotype, which has emerged as an important serotype infecting humans recently, presence of G9 serotype was expected in nontypeable specimens. Therefore, we selected specimens from those children, who showed higher neutralizing antibody (NAb) titer in their convalescent serum samples to G9 serotype than their mothers. Out of six isolates having long electropherotype, five isolates showed subgroup II, and one showed subgroup I, II. The isolates were confirmed as G9 by MAb based ELISA, neutralization assay, and PCR. The G9 specific nested PCR products of four isolates showed 96-99% identities to AU32 G9 strain reported from Japan. P type of four isolates was determined as P8. Besides isolates, four additional nontypeable fecal specimens were confirmed as G9 by MAb based ELISA. Thus, 10 (28.57%) out of 35 nontypeable specimens were identified as rotavirus serotype G9. The results indicate that serotype G9 may represent significant proportion of specimens, which were previously nontypeable.     

Rapid identification of staphylococcal strains from positive-testing blood culture bottles by internal transcribed spacer PCR followed by microchip gel electrophoresis

PCR analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) followed by microchip gel electrophoresis (MGE) was evaluated for its usefulness in identification of staphylococci. Forty ITS PCR patterns were demonstrated among 228 isolated colonies of Staphylococcus aureus: 26 patterns for methicillin-susceptible S. aureus (MSSA; 91 strains), 11 patterns for methicillin-resistant S. aureus (MRSA; 99 strains), and 3 patterns for both MSSA and MRSA (38 strains). Thirty-seven control strains of coagulase-negative staphylococci (CNS) representing 16 species showed unique ITS PCR patterns (24 patterns) at the species and subspecies levels: two patterns for S. caprae, S. cohnii, S. haemolyticus, and S. saprophyticus; three patterns for S. lugdunensis; four patterns for S. capitis; and one pattern for each of the other CNS species. The combined PCR-MGE method was prospectively adapted to the positive blood culture bottles, and this method correctly identified MSSA and MRSA in 102 (89%) of 114 blood cultures positive for S. aureus on the basis of the ITS PCR patterns. Eight ITS PCR patterns were demonstrated from 166 blood culture bottles positive for CNS. The most frequent CNS species isolated from blood cultures were S. epidermidis (76%), S. capitis (11%), and S. hominis (8%). Overall, all 280 blood culture bottles shown to contain a single Staphylococcus species by routine phenotypic methods were correctly identified by the PCR-MGE method at the species level, whereas the organism failed to be identified in 8 culture bottles (3%) with mixed flora. The PCR-MGE method is useful not only for rapid identification ( approximately 1.5 h) of staphylococci in positive blood culture bottles, but also for strain delineation of S. aureus.     

Detection of superficial zone protein in human and animal body fluids by cross-species monoclonal antibodies specific to superficial zone protein.

In this report we describe the purification of human superficial zone protein (SZP), the generation of cross-species monoclonal antibodies (MAbs) and the detection of this protein in human and animal body fluids. Human SZPs, used as immunizing antigens, were purified either from culture media of human cartilage organ cultures or from human synovial fluids. The immunizing antigens were mixed with RIBI adjuvant in one of three forms: nonmodified SZP, superficial zone protein-keyhole limpet hemocyanin conjugate (SZP-KLH), or a mixture of superficial zone protein and hyaluronic acid (SZP-HA). A panel of MAbs including GW4.23, S6.79, S13.52, S13.233, and S17.109 were generated and characterized. Monoclonal antibody (MAb) S6.79, an IgG2b with K(D) 3.14 x 10(-9) M from SZP-KLH immunization, is of particular interest. It reacts strongly to a large molecular weight form of SZP in both enzyme-linked immunosorbent assay (ELISA) and Western blotting. It stains the most superficial layer of articular cartilage in immunohistochemistry, whereas the middle and deep zones of cartilage are not stained. When MAb S6.79 was applied to Western blots of human body fluids, a strong 345-kDa band was detected in samples of synovial fluid and weaker bands of similar size were detected in samples of plasma and serum. MAb S6.79 also showed cross-species immunoreactivity with SZP in samples of synovial fluids harvested from bovine, dog, guinea pig, and rabbit, as demonstrated by Western blotting and antibody absorption experiments. This cross-species MAb will be a useful tool in human and animal model studies for monitoring SZP levels and tissue distribution. It may help define the roles of SZP in normal articular joints and may be of diagnostic or prognostic value for the measurement of SZP in pathological conditions such as osteoarthritis, rheumatoid arthritis, and camptodactyly-arthropathy-coxa vara-pericarditis.     

Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein-labeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy.     

Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes

The applicability of whole-cell hybridization for the identification of pathogenic bacteria in blood from septic patients was examined. Oligonucleotide probes, fluorescently labeled with fluorescein isothiocyanate, directed against the variable regions of the 16S rRNAs of the following bacterial species and/or genera were used: Streptococcus spp., Enterococcus faecalis, Staphylococcus aureus, coagulase-negative staphylococci (CoNS), Escherichia coli, Pseudomonas aeruginosa, and the Enterobacteriaceae family. A probe specific for the rRNAs of almost all bacteria and its complementary, reversed counterpart was used as positive and negative control, respectively. The probes were used in conjunction with a fast and simple-to-use protocol for whole-cell hybridization. This protocol yields an identification after 25 to 45 min, depending on whether the bacterium is gram positive or gram negative. A total of 182 blood samples which tested positive in a blood culture machine were investigated. All probes except for the ones for S. aureus and the CoNS showed sensitivities and specificities of 1.000. It was concluded that whole-cell hybridization is well suited for the fast screening of septic blood containing streptococci and/or enterococci or gram-negative rods.     

Comparative studies of the antigenic polypeptide species VP4, VP6, and VP7 of three strains of bovine rotavirus.

Three bovine rotavirus strains belonging to two distinct serotype groups, serotype 6 (NCDV and B641) and B223, distinct from the other six mammalian rotavirus serotypes but not yet assigned to a serotype group, were compared with each other and with canine rotavirus (K9, serotype 3) by studying the properties of their cognate polypeptide species VP4, VP6, and VP7. The three viruses showed distinct differences in the polyacrylamide gel electrophoretic migration rates of protein species VP4 and VP7, with minor differences in VP6. Differences were also observed among the migration patterns of genome segments 4, 6, and the 7-8-9 triplet, which encode VP4, VP6, and VP7, respectively. Monoclonal antibodies (MAbs) to B223, which were directed against VP4 or VP7, showed homologous specificity for neutralization and immunofluorescence (IF), although one MAb reactive with VP4 also reacted by IF and by immunoprecipitation (IP) with all four viruses and weakly neutralized B641 and K9. This MAb may react with the epitope responsible for the B223-induced one-way neutralizing and protection response of calves against B641 observed in earlier studies. MAbs reactive with VP6 by IP showed enzyme-linked immunosorbent assay and IF reactivity with all three bovine viruses and the canine virus. The two serotype 6 viruses could be distinguished by the two B641 MAbs, B641-N2b reacting by neutralization and IF with both viruses and B641-N1 reacting with B641 and the serotype 3 canine rotavirus but not with NCDV. One nonneutralizing B641 MAb reacted by IP and IF with VP7 of all four rotaviruses examined, and one B223 MAb neutralized B223 and, to low titer, B641 and K9 although reacting by IP and IF with all four viruses. Three MAb-resistant mutants were selected by passage of B223 in the presence of one of three selected B223 MAbs at concentrations which only neutralized approximately 90% of the infectious virions. The resulting mutants were 100% resistant to neutralization with their respective MAb but remained neutralizable by the same selection of MAbs as the parent B223 virus.