An application of monoclonal antibodies to identification of leptospires of serogroup icterohaemorrhagiae found in China

For the purpose of improving the procedures of identification of leptospires, a set of 5 monoclonal antibodies with different serological reactivity against serovars of Leptospira interrogans Icterohaemorrhagiae serogroup isolated in China was developed. One hundred and eight strains isolated from epidemic fields in 5 provinces in southern China were distinctly identified into 4 serovars of Icterohaemorrhagiae serogroup by the monoclonal antibody procedure, i.e., 98 isolates were identified as serovar lai, 7 as icterohaemorrhagiae, 2 as copenhageni, and 1 as H2. Factor antiserum procedure was used at the same time as control for typing these strains and an identical result was obtained.     

Serological analysis of H-2 and Ia molecules with monoclonal antibodies

Five H-2 and seven Ia monoclonal antibodies were tested against a panel of 43 independentH-2 haplotypes (11 of laboratory-mouse and 32 of wild-mouse origin), 33 recombinantH-2 haplotypes, and up to 74 wild mice. All the antibodies gave negative reactions in the PVP hemagglutination tests; all, however, gave positive reaction with some members of the panel in the dye-exclusion cytotoxic test. Four of the antibodies (H-2.m2,Ia.m2, Ia.m5 and Ia.m7) reacted identically to conventional antibodies detecting determinants H-2.2., and Ia-1.2, Ia-1.15, and Ia-5.7, respectively (this statement does not apply to wild mice in which minor differences in reactivity patterns of the corresponding antibodies were found: the reproducibility of these differences, however, could not be checked by absorption). Five other antibodies (H-2.m5, H-2.m3, H-2.m4, Ia.ml, and Ia.m6) had very similar though not identical reactivity patterns to conventional antibodies detecting determinants H-2.5, H-2.11, H-2.25, Ia-1.2, and Ia-1.19, respectively. The last three monoclonal antibodies (H-2.ml, Ia.m3, and Ia.m4) had a reactivity pattern that did not match those of any known conventional antibodies. The near identity or great similarity of many monoclonal and conventional antibodies indicates that the cleanest of the conventional antisera are truly monospecific, and gives credence to the H-2 serology as defined by conventional antibodies. The serological analysis of monoclonal antibodies supports the true existence of private and public determinants, and reveals that the H-2 and Ia determinants are complex, even when the antibody is simple.     

Two murine monoclonal antibodies against serogroup E Salmonellae

A monoclonal antibody (MAb), MO15, was raised against the lipopolysaccharide antigen of an epsilon15-lysogenized serogroup E(1) Salmonella strain. The O factor 15-specific MAb MO15, together with another serogroup E-specific MAb, can differentiate among phage lysogenization variants in serogroup E salmonellae. Their epitope specificities in relation to conventional O-antigenic structures are discussed.     

Production of monoclonal antibodies against Legionella pneumophila serogroup 1

Four monoclonal antibodies to Legionella pneumophila Philadelphia 1 were produced by the fusion of immunized BALB/c lymphocytes to a murine myeloma cell line. Two (Lp1-1 and Lp1-3) of the four monoclonal antibodies reacted with 14 L. pneumophila serogroup 1 strains, and the other (Lp1-2 and Lp1-4) reacted with only three out of 20 strains tested. These four monoclonal antibodies did not bind to any strains of L. pneumophila serogroup 2-7 and other Legionella species. In addition, it has been shown that these monoclonal antibodies may be useful not only for subserotyping of L. pneumophila but also for retrospective diagnosis using immunopathological methods.     

Serological studies on Leptospira strain Ictero No. I using monoclonal antibodies.

In order to elucidate the full serological characteristics of strain Ictero No. I, the first strain of Leptospira isolated by Inada and Ido in 1914, 17 monoclonal antibodies against the strain were produced by cell fusion technology. The antibody-producing hybridomas were designated IMAs 1 to 17. The reactivities of the monoclonal antibodies produced by the hybridomas were determined by the microscopic agglutination test. One of the 17 monoclonal antibodies, IMA 1, reacted with strains Ictero No. I, LT 1131 and Naam, but not with other strains of the serogroup Icterohaemorrhagiae including strain RGA used in the present study. Moreover, the reactivity of the antigenic determinant of IMA 1 was inactivated by treatment at 56 C for 30 min. The 16 other monoclonal antibodies, IMAs 2 to 17, showed different reaction patterns against Leptospira strains of the serogroup Icterohaemorrhagiae. All of the 16 antibodies reacted with both Ictero No. I and RGA strains. The antigens against antibodies IMAs 2 to 17 were thermostable. The present study thus clarified the presence of a thermolabile antigen in strain Ictero No. I, and allowed correct distinction between the serotype of strain Ictero No. I and that of strain RGA using IMA 1. Therefore, we propose that strain Ictero No. I represents serovar icterohaemorrhagiae, as originally designated by Inada and Ido. Moreover, strain Ictero No. I should be designated as the type strain of Leptospira interrogans.     

Functional analysis of TRAIL receptors using monoclonal antibodies

mAbs were generated against the extracellular domain of the four known TNF-related apoptosis-inducing ligand (TRAIL) receptors and tested on a panel of human melanoma cell lines. The specificity of the mAb permitted a precise evaluation of the TRAIL receptors that induce apoptosis (TRAIL-R1 and -R2) compared with the TRAIL receptors that potentially regulate TRAIL-mediated apoptosis (TRAIL-R3 and -R4). Immobilized anti-TRAIL-R1 or -R2 mAbs were cytotoxic to TRAIL-sensitive tumor cells, whereas tumor cells resistant to recombinant TRAIL were also resistant to these mAbs and only became sensitive when cultured with actinomycin D. The anti-TRAIL-R1 and -R2 mAb-induced death was characterized by the activation of intracellular caspases, which could be blocked by carbobenzyloxy-Val-Ala-Asp (OMe) fluoromethyl ketone (zVAD-fmk) and carbobenzyloxy-Ile-Glu(OMe)-Thr-Asp (OMe) fluoromethyl ketone (zIETD-fmk). When used in solution, one of the anti-TRAIL-R2 mAbs was capable of blocking leucine zipper-human TRAIL binding to TRAIL-R2-expressing cells and prevented TRAIL-induced death of these cells, whereas two of the anti-TRAIL-R1 mAbs could inhibit leucine zipper-human TRAIL binding to TRAIL-R1:Fc. Furthermore, use of the blocking anti-TRAIL-R2 mAb allowed us to demonstrate that the signals transduced through either TRAIL-R1 or TRAIL-R2 were necessary and sufficient to mediate cell death. In contrast, the expression of TRAIL-R3 or TRAIL-R4 did not appear to be a significant factor in determining the resistance or sensitivity of these tumor target cells to the effects of TRAIL.     

Serological study of yeast killer toxins by monoclonal antibodies

Yeast killer toxins coded by determined and undetermined killer plasmids or presumptive nuclear gene(s) in various genera (Saccharomyces, Kluyveromyces, Pichia and Candida) have been serologically investigated by a monoclonal antibody (KT4), produced against the yeast killer toxin of Pichia (Hansenula) anomala UCSC 25F. Double immunodiffusion with the killer toxins as antigens and indirect immunofluorescence on whole cells of the corresponding killer yeast have been used. In both the serological procedures, monoclonal antibody KT4 proved to be reacting only with the killer toxins and the whole cells of yeasts belonging to the genus Pichia.     

Antigenic analysis of testicular cytochromes c using monoclonal antibodies

Testicular cytochrome c (cyt ct) was isolated from testes of sexually mature, rat, mouse, rabbit, and bull, among which rat testis is highly rich in cyt ct. By fusion of NS-1 myeloma cells and spleen cells of mice immunized with rat cyt ct, 11 stable mouse hybridoma cell lines were established. Using an enzyme-linked immunosorbent assay, it was determined that 4 of the 11 anti-rat cyt ct monoclonal antibodies (McAb) did not bind to somatic cyt c (cyt cs) of vertebrates nor to cyt ct of mouse, rabbit, and bull. Four other McAb showed no binding to cyt cs but showed different patterns of cross-reactivity with these four cyt ct. Therefore, these McAb appear to be very sensitive and useful probes for the discrimination or identification of extremely similar isocytochromes c. Although the primary amino acid sequences between cyt cs of rat and mouse are identical, the antigenic structure of cyt ct of rat and mouse are clearly distinct with regard to cross-reactivity with some anti-rat cyt ct McAb. Furthermore, these McAb also reveal that the primary amino acid sequences of cyt ct, which reflect differences in the surface conformation of the molecule, are probably species specific.     

Serological analysis of early mouse embryo with rat monoclonal antibodies produced against mouse teratocarcinoma cells

Rat-mouse hybridoma antibodies were produced against mouse teratocarcinoma F9 or PCC4 aza1 cells, and four clones were established. Both the F11 (IgM) and F20 (IgG2c) antibodies showed a similar specificity, reacting only with nullipotential teratocarcinoma cells. They were also found to agglutinate sheep red blood cells. Solid-phase enzyme-linked immunofluorescence assay showed that, among the neutral glycolipids studied, they only reacted with the Forssman antigen. P2 antibody (IgG2b) reacted with the undifferentiated-type and embryonal endodermtype teratocarcinoma cells. During the preimplantation stage, this antibody did not stain mouse embryos, but it reacted very weakly with the inner cell mass of blastocysts cultured in vitro. In the 5th-day embryo, the embryonic ectoderm as well as the visceral and parietal endoderm were positive, but the extraembryonic ectoderm was not. Mesoderm of the 7.5th-day embryo also reacted with this antibody. However, P2 antigen was not observed in the 16th-day embryo or in adult tissues. F2 antibody (IgG2a), which was reactive with all of the cultured cell lines tested, showed an immunoreaction with mouse embryos throughout the preimplantation stage. However, in the 7.5th-day embryo, the presence of F2 was limited to the cells forming the parietal endoderm. This antigen was present in some epithelial tissues of the 16th-day embryo and adult mouse. Of these antigens, P2 and F2 are probably novel differentiation antigens of the early mouse embryo. Together with the Forssman antigen, these will be important markers for analyzing cell-surface antigens of mouse teratocarcinoma cells as well as embryos.     

Desmin-containing intermediate filaments: structural analysis using monoclonal antibodies

1. Monoclonal antibodies have been raised against N- and C-terminal desmin sequence regions. 2. The antibodies decorated desmin intermediate filaments in a helical fashion with four antibody molecules per helix turn. 3. The filaments could be decorated with both types of antibody consecutively. 4. These results support a model of intermediate filament assembly in which the tetrameric protofilaments are aligned in a staggered fashion with partial overlapping of the central rod domain regions of the desmin sequence.     

The immunofluorescent detection of Leptospira interrogans serogroup Icterohaemorrhagiae serovar icterohaemorrhagiae infections in human tissues

The identification of Leptospira interrogans serogroup Icterohaemorrhagiae serovar icterohaemorrhagiae infections in post-mortem tissues by direct fluorescence microscopy is demonstrated by the use of a serogroup-specific fluorescein isothiocyanate label.     

Phylogenetic study of transcortin using monoclonal antibodies

We produced monoclonal antibodies that recognise three distinct epitopes of human transcortin. These epitopes are present on transcortin of humans with normal and altered transcortin levels, as well as on a variant with lower affinity for cortisol. One epitope is present on transcortin of Old World Monkeys and apes, the others are only present on transcortin of apes. The epitopes are not present on transcortin of other species. These results indicate that human transcortin contains a highly evolved and a more conserved part.     

Characterization of syngeneic rat monoclonal antibodies to the HSN tumor using syngeneic monoclonal anti-idiotopic antibodies

Twelve syngeneic anti-idiotopic mAb (anti-idiotypic/idiotopic antibodies Ab2)) were prepared from CBH/Cbi rats immunized with one of three monoclonal anti-HSN antibodies (Ab1) (11/160, ALN/11/53, or ALN/16/53) specific for the HSN tumor. The sera of the rats used for hybridoma production and all of the monoclonal Ab2 specifically inhibited the binding to HSN of the immunizing Ab1 only. It is concluded that, in this completely syngeneic system, only the private idiotopes associated with the antibody-combining site were immunogenic. Analyses using Western blotting showed that the Ab2 bound to intact Ab1 and to isolated H chains where the intra-strand disulfide bonds remained intact. The Ab2 did not bind to L chains or to fully reduced H chains of the Ab1. It is concluded that the idiotopes expressed on the H chain were conformational. When a panel of 13 monoclonal Ab1 (including the three used for immunization) were tested for reactivity with the Ab2, three reacted specifically with their respective Ab2 and 8 gave no binding suggesting that each Ab1 had a distinct idiotypic specificity despite the fact that all the Ab1 competed with each other for binding to Ag. However, the two remaining Ab1 (ALN/9/94 and ALN/12/17) generated from different tumor-bearing rats, were found to possess the same idiotypic specificity as 11/160. A detailed analysis using seven Ab2 raised against 11/160 showed that while the idiotype of ALN/9/94 and 11/160 were very similar, that of ALN/12/17 showed some clear differences. These three Ab1 have been shown previously to bind a sequential epitope on the HSN Ag in Western blots and it is postulated that the common idiotype of these Ab1 reflects their recognition of a sequential epitope. This may also account for the relatively frequent occurrence in tumor bearer sera of antibodies with this Id.     

Serological characterization of potato isolates of Erwinia carotovora subsp. atroseptica and subsp. carotovora using polyclonal and monoclonal antibodies

Serological, biochemical and physiological characteristics of 81 strains of Erwinia carotovora subsp. atroseptica ( Eca ) and 67 strains of subsp. carotovora ( Ecc ) from potato, isolated in Spain and from several international collections, have been studied. Ouchterlony double diffusion (ODD), indirect immunofluorescence (IIF) and indirect enzyme-linked immunosorbent assay (ELISA) were the methods used. The antibodies were polyclonals from eight antisera prepared with Eca serogroup I and Ecc serogroup III and two monoclonal antibodies (MAbs), 4G4 from Spain and 4F6 from Canada, both prepared with Eca strains of serogroup I. Serogroup I for Eca and several serogroups for Ecc were the most commonly found in the collection studied. Serological relationships between Eca and Ecc independently of the serogroups were observed by IIF and ELISA using polyclonal antibodies. Common epitopes between all Eca and Ecc studied were detected. Both MAbs recognized epitopes in Eca strains of serogroups I and XXII in IIF and ELISA but they did not react with strains of other serogroups nor Ecc strains. The pattern of reaction against the strains assayed was rather similar but not identical indicating that they represent two different and well conserved epitopes. This study confirms the serological complexity of Ecc and Eca and gives information about the serological probes for detection of both subspecies.     

Topological analysis of the Escherichia coli ferrichrome-iron receptor by using monoclonal antibodies.

Ferrichrome-iron transport in Escherichia coli is initiated by the outer membrane receptor FhuA. Thirty-five anti-FhuA monoclonal antibodies (MAbs) were isolated to examine the surface accessibility of FhuA sequences and their contribution to ligand binding. The determinants of 32 of the MAbs were mapped to eight distinct regions in the primary sequence of FhuA by immunoblotting against (i) five internal deletion FhuA proteins and (ii) four FhuA peptides generated by cyanogen bromide cleavage. Two groups of MAbs bound to FhuA in outer membrane vesicles but not to intact cells, indicating that their determinants, located between residues 1 and 20 and 21 and 59, are exposed to the periplasm. One of the 28 strongly immunoblot-reactive MAbs bound to FhuA on intact cells in flow cytometry, indicating that its determinant, located between amino acids 321 and 381, is cell surface exposed. This MAb and four others which in flow cytometry bound to cells expressing FhuA were tested for the ability to block ligand binding. While no MAb inhibited growth promotion by ferrichrome or cell killing by microcin 25, some prevented killing by colicin M and were partially able to inhibit the inactivation of T5 phage. These data provide evidence for spatially distinct ligand binding sites on FhuA. The lack of surface reactivity of most of the immunoblot-reactive MAbs suggests that the majority of FhuA sequences which lie external to the outer membrane may adopt a tightly ordered organization with little accessible linear sequence.     

Inter-specific analysis of Drosophila alcohol dehydrogenase by an immunoenzymatic assay using monoclonal antibodies

Three Drosophila alcohol dehydrogenase monoclonal antibodies have been prepared and characterized. These antibodies cross-react with alcohol dehydrogenase from different species as revealed by immunoblotting assay. An enzyme-linked immunosorbent assay has been devised to quantify alcohol dehydrogenase in several species, different strains and individual larval organs. The assay detects alcohol dehydrogenase via a double-antibody sandwich assay technique giving strictly proportional values for antigen concentration and optical densities in the range of 3-30 ng of antigen per 100 microliters of sample. When alcohol dehydrogenase specific activity is compared in different larval organs a remarkable similarity is observed, whereas protein distribution varies substantially. Larval fat body and larval alimentary canal contribute 63% and 26% respectively to recovered alcohol dehydrogenase.     

Serological evidence of California serogroup virus activity in Oregon

We wished to demonstrate evidence of the presence of California serogroup viruses in Oregon and to test for the presence of certain other arboviruses in large ungulates. Blood samples from black-tailed deer (Odocoileus hemionus columbianus), mule deer (O. hemionus hemionus), and Roosevelt elk (Cervus elaphus roosevelti) from nine counties in Oregon were tested by serum-dilution plaque reduction neutralization for antibody to California serogroup viruses, including snowshoe hare, California encephalitis, and Jamestown Canyon, as well as to Cache Valley (Bunyamwera serogroup) and Klamath, an ungrouped rhabdovirus. Of 132 samples tested, 60 (46%) were found to be seropositive at a dilution of greater than or equal to 1:10 for at least one of the five different arboviruses. Forty (30%) samples contained antibody to more than one arbovirus, and 15 samples (11%) contained antibody to all five. Of these 15, 14 were from 75 black-tailed deer sera collected in Lincoln County, Oregon. Seropositivity rates for black-tailed deer ranged from 23% to 35%, with all five arboviruses represented. Positive reactions for all five arboviruses were represented among mule deer sera at rates from 5% to 29%. Elk sera were found to be positive for four of the viruses (none for Klamath virus). Although Cache Valley and Klamath viruses have been reported from Oregon, these data represent the first evidence of a California serogroup virus in the state.