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PubMed1992

Development and characterization of a panel of monoclonal antibodies against the novel subtilisin-like proprotein processing enzyme furin.

Monoclonal antibodies were raised against the recently discovered subtilisin-like proprotein processing enzyme furin. As immunogen, a bacterially expressed hybrid protein was used which consisted of glutathione S-transferase fused to almost the entire human furin protein. Ten monoclonal antibodies were obtained and these could be divided into four categories on the basis of their reactivity towards a number of bacterially expressed hybrid proteins, each of which contained a different portion of human furin. Four of the monoclonal antibodies did not recognize mouse furin. All monoclonal antibodies were tested for their applicability in Western blot and immunofluorescence analysis. Western blot analysis was performed with COS-1 cells in which biologically active forms of human and mouse furin were expressed transiently under control of the SV40 late promoter. This approach was necessary, since physiological levels of fur gene encoded proteins appeared to be very low. In cells transfected with human or mouse fur cDNA, a protein of about 100 kDa and a doublet of about 90 kDa could be detected with most of the monoclonal antibodies. Some of these antibodies appeared to be also reactive in immunofluorescence analysis of transfected COS-1 cells.

Animals↗
PubMed1990

Laminin receptors on Candida albicans germ tubes.

Recent evidence for the role of laminin in cell adhesion and in the pathogenesis of several bacterial infections has led us to investigate the existence of receptors for this extracellular matrix component in Candida albicans. At first, immunofluorescence demonstrated the presence of laminin-binding sites at the surface of germ tubes. Electron microscopy confirmed this result and permitted precise localization of the binding sites on the outermost fibrillar layer of the germ tube cell wall. By using 125I-radiolabeled laminin, the binding was shown to be saturable and specific, hence demonstrating characteristics of true receptors. Analysis of the data by the Scatchard equation indicated that there were about 8,000 binding sites per cell, with a dissociation constant (Kd) of 1.3 x 10(-9) M. Binding was inhibited by prior heating or trypsinization of cells. Furthermore, of the different proteins and carbohydrates tested in competition experiments, only fibrinogen greatly reduced the laminin binding. Finally, dithiothreitol and iodoacetamide treatment of germ tubes allowed us to identify the laminin receptors through analysis of this extract by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. Two components, of 68 kilodaltons and a doublet of 60 and 62 kilodaltons, were detected. Thus, C. albicans possesses germ tube-specific surface receptors for laminin which could mediate its attachment to basement membranes and so contribute to the establishment of candidiasis.

Candida albicans↗
PubMed1997

Organization of protein and mRNA for titin and other myofibril components during myofibrillogenesis in cultured chicken skeletal muscle.

Myofibril assembly requires the cell to join diverse components, correctly oriented to the rest of the cell. Titin, a huge elastic protein with a role in myogenesis, assembles during translation in vivo and may require spatially organized mRNA to allow assembly. By immunofluorescence, we examined titin and myosin protein organization early in skeletal muscle development in vitro; titin was the first organized, initially as spots, then as periodically spaced lines, and later as doublets. Titin mRNA organization during development was detected by fluorescent in situ hybridization. Only titin mRNA was seen in mononucleated myoblasts. Shortly after fusion, both titin protein and mRNA were diffuse. Titin mRNA remained diffuse when titin protein formed cables. Where titin protein formed linear arrays of spots, titin mRNA showed a colinear, continuous array. Titin mRNA remained in arrays colinear with young myofibrils until several slender myofibrils aligned laterally; then, titin mRNA formed periodic arrays. The titin probe encodes peptide sequence in the A band, where this region of titin mRNA is detected in the most organized cells. Nebulin undergoes a similar progression slightly later in development. This pattern, of narrowly spaced stripes, is too closely spaced to function in the soluble phase. Titin mRNA is the earliest mRNA to become so highly organized in muscle; it does so earlier and at a different location than do mRNAs for costamere proteins. These results, taken with earlier ones, suggest mRNA localization may be as key to somatic cell differentiation as it is to embryonic development.

Animals↗
PubMed1992

A unique Kex2-like endoprotease from Drosophila melanogaster is expressed in the central nervous system during early embryogenesis.

Complementary DNA sequences were cloned from a Drosophila library encoding a 1,101 amino acid polypeptide that we have named dKLIP-1. The deduced protein is structurally similar to the yeast KEX2 prohormone endoprotease including the conserved Asp, His, and Ser catalytic triad residues characteristic of the subtilisin family. When coexpressed in vivo with pro-beta-NGF, dKLIP-1 greatly enhanced the endoproteolytic conversion of the precursor to mature beta-NGF by cleavage at a -Lys-Arg- doublet. In adults, dKLIP-1 transcripts were detected in cortical regions of the CNS and fat body. Most striking, however, was the high level of maternal transcripts deposited into developing oocytes. The temporal and spatial expression of dKLIP-1 mRNAs during embryonic development indicates a potential role for this novel Kex2p-like endoprotease in early embryogenesis and neurogenesis.

Animals↗
PubMed1990

Assembly and activation of the NADPH:O2 oxidoreductase in human neutrophils after stimulation with phorbol myristate acetate.

Phagocytic leukocytes contain an activatable NADPH:O2 oxidoreductase. Components of this enzyme system include cytochrome b558, and three soluble oxidase components (SOC I, SOC II, and SOC III) found in the cytosol of resting cells. Previously, we found that SOC II copurifies with, and is probably identical to, a 47-kDa substrate of protein kinase C. In the present study we investigated the change in location of several of these oxidase components after activation of intact neutrophils with phorbol myristate acetate (PMA) and separation of subcellular fraction on sucrose density gradients. On Western blots with fractions of resting cells, the alpha subunit of cytochrome b558 was detected with a monoclonal antibody as a doublet of Mr 22,000 and 24,000 in the specific granules and as a single band of Mr 24,000 in the plasma membrane. PMA induced an increase of cytochrome b558 in the plasma membrane, including the Mr 22,000 band. PMA also induced translocation of the 47-kDa protein from the cytosol to the membrane fraction, as revealed by in vitro phosphorylation experiments. When NADPH oxidase activity was determined in a cell-free system in the presence of sodium dodecyl sulfate and GTP with plasma membranes from resting cells, cytosol from PMA-treated cells was deficient compared with cytosol from resting cells. This deficiency could be partially restored by the addition of SOC I. Concomitantly, SOC I activity appeared in the plasma membranes of PMA-treated cells. These studies support the hypothesis that PMA stimulation of neutrophils results in assembly of oxidase components from the cytosol and the specific granules in the plasma membrane with subsequent expression of NADPH oxidase activity.

Antibodies, Monoclonal↗
PubMed1979

Heterogeneity in cell-associated transformation-sensitive proteins antigenically related to fibronectin.

Antiserum to purified fibronectin has been used to investigate transformation-associated heterogeneity in surface components antigenically related to fibronectin. Examination of extracts from surface-labeled rat cells by gradient gel electrophoresis revealed in "normal" cells the presence of two species with approximate molecular weights of 250,000 and 230,000, which were decreased in wild-type transformed cells. Reaction with antifibronectin serum revealed the selective precipitation of the two transformation-sensitive surface components. A similar experiment with ts-NT3-KR that expresses a normal phenotype at 37 degrees and a transformed morphology at 33 degrees did not reveal a markedly altered surface labeling pattern at both temperatures. However, reaction with antifibronectin serum did show a weak but detectable recognition of a 230,000-dalton doublet in the cells grown at 37 degrees, and immune precipitation of components in the 100,000- and 60,000-dalton region in cells grown at 33 degrees. Experiments with normal mouse cells revealed a different ratio of two fibronectin-related external proteins with similar molecular weights to those seen in normal rat cells.

Animals↗
PubMed1989

Detection of GTP-binding proteins in purified derivatives of rough endoplasmic reticulum.

As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alpha-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [alpha-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types.

Animals↗
PubMed1999

Composition and organization of tubulin isoforms reveals a variety of axonemal models.

In the flagellum of mammalian spermatozoa, glutamylated and glycylated tubulin isoforms are detected according to longitudinal gradients and preferentially in axonemal doublets 1-5-6 and 3-8, respectively. This suggested a role for these tubulin isoforms in the regulation of flagellar beating. In the present work, using antibodies directed against various tubulin isoforms and quantitative immunogold analysis, we aimed at investigating whether the particular accessibility of tubulin isoforms in the mammalian sperm flagellum is restricted to this model of axoneme surrounded with periaxonemal structures or is also displayed in naked axonemes. In rodent lung ciliated cells, all studied tubulin isoforms are uniformly distributed in all axonemal microtubules with a unique deficiency of glutamylated tubulin in the transitional region. A similar distribution of tubulin isoforms is observed in cilia of Paramecium, except for a decreasing gradient of glutamylated tubulin labeling in the proximal part of axonemal microtubules. In the sea urchin sperm flagellum, predominant labeling of tyrosinated and detyrosinated tubulin in 1-5-6 and 3-8 doublets, respectively, were observed together with decreasing proximo-distal gradients of glutamylated and polyglycylated tubulin labeling and an increasing gradient of monoglycylated tubulin labeling. In flagella of Chlamydomonas, the glutamylated and glycylated tubulin isoforms are detected at low levels. Our results show a specific composition and organization of tubulin isoforms in different models of cilia and flagella, suggesting various models of functional organization and beating regulation of the axoneme.

Animals↗
PubMed2000

Effects of epinephrine infusion on expression of calpastatin in porcine cardiac and skeletal muscle.

beta-Adrenergic agonists induce muscle hypertrophy in mammalian species and alter the extractable activity of calpain proteinase and its specific endogenous inhibitor calpastatin. The effects on skeletal and cardiac muscle calpastatin of continuously infusing a group of pigs for 7 days with the physiological agonist epinephrine (0.15 microg/kg/min) were examined and compared with a placebo group. Basal levels of extractable calpastatin activity were higher in cardiac than skeletal muscle and epinephrine infusion increased the extractable activity in both muscle types (P < 0.05). An anti-recombinant porcine calpastatin antiserum detected a 135-kDa band and a 145/135-kDa doublet on Western blots of skeletal and cardiac extracts, respectively. Epinephrine infusion increased the 135-kDa band in skeletal muscle (P < 0.05), while the ratio of 145/135 kDa in cardiac muscle was decreased (P < 0.05). From Northern blots, the patterns of calpastatin mRNA species were similar in the two muscle types, two major transcripts at 5.8 and 3. 2 kb in cardiac muscle, with equivalent bands in skeletal muscle of 5.4 and 2.8 kb. A faint 7.9-kb band was also detected in skeletal muscle. Epinephrine infusion had no effect on skeletal calpastatin mRNA but tended to increase the 5.8-kb mRNA in cardiac muscle (P = 0. 053). These data indicate a differential response of the two muscle types to mildly elevated plasma epinephrine concentration and the response to elevated epinephrine may be at the translational or posttranslational level. Therefore, this type of stimulus appears to be less effective at perturbing calpastatin gene transcription than certain orally administered synthetic beta-adrenergic agonists.

Animals↗
PubMed1991

Blood-borne fragments of fibronectin after thermal injury.

Fibronectin is an adhesive protein that can promote phagocytosis and endothelial cell adhesion. Plasma fibronectin declines following burn in animals and patients, potentially due to its complexing with circulating collagenous debris as well as its rapid binding to sites of tissue injury. Such depletion of fibronectin initiates an opsonic deficiency of the plasma. In view of the sensitivity of fibronectin to proteolytic enzymes, an additional factor that could contribute to the decrease of plasma opsonic activity after burn is the proteolytic fragmentation of fibronectin in the blood. In the current study, we determined if fibronectin fragments appear in the blood of anesthetized rats after a sublethal full-thickness skin burn of 15% to 16% of body surface. Plasma fibronectin concentration was quantified by enzyme-linked immunosorbent assay and the presence of fibronectin fragments in plasma was determined by immunoblot analysis. All blood was collected in an antiprotease mixture to yield final plasma concentrations of 0.15% EDTA, 3mmol/L phenylmethylsulfonyl fluoride, and 3 mmol/L iodoacetate to prevent degradation of fibronectin after sampling. Plasma fibronectin decreased 60% to 70% within 30 minutes post-burn, and this low level lasted for at least 4 hours. Within 30 minutes post-burn, two prominent fragments of fibronectin with a molecular weight of 110 +/- 2.2 kd and 122 +/- 3.3 Kd, respectively, were also detected in the plasma. Peak concentration of these fragments was detected at 60 minutes post-burn, but their level declined by 4 hours. By 4 hours, both bands appeared to resolve into doublets. To rule out the possibility that the fragments of fibronectin detected in the plasma were actually generated by coagulation enzymes activated at the site of peripheral blood sampling, rapid direct inferior vena cava sampling was performed, which also yield the presence of the fragments. Thus, fibronectin fragments exist in the plasma following thermal injury. Because fragments of fibronectin can compete with the intact fibronectin molecule with respect to its ability to stimulate macrophage phagocytosis, such fragments may contribute to altered systemic phagocytic host defense following thermal injury. Furthermore, because fibronectin peptides can compete with matrix fibronectin and impair adhesion of cultured endothelial cells, such circulating fragments may also influence the integrity of the vascular barrier.

Animals↗
PubMed1995

Direct detection of ascorbyl radical in experimental brain injury: microdialysis and an electron spin resonance spectroscopic study.

To examine the role played by free radicals in brain injury, we performed experiments to detect radicals in the frontal cortex of rats, using electron spin resonance (ESR) and microdialysis. A dialysis probe was inserted into the frontal cortex, and spin adducts in perfusates were immediately detected by ESR. We obtained a relatively stable doublet signal, with parameters of g = 2.0057 and aH = 0.17 mT. This signal corresponded with that of the ascorbyl radical. Ascorbyl radical in the perfusate collected from the frontal cortex was augmented by microinjection of H2O2 and FeCl2 adjacent to the dialysis probe. When the rats were challenged with cold-induced brain injury, ascorbyl radical and lactate dehydrogenase (LDH) level in the perfusate increased significantly. Pretreatment with superoxide dismutase and catalase attenuated the increase in ascorbyl radical and LDH level induced by the cold injury. Infusion of FeCl2 dissolved in perfusate caused a pronounced increase in ascorbyl radical and LDH level after the cold injury. We conclude that the direct detection of free radical formation further supports the hypothesis that free radicals play an important role in traumatic brain injury. Our findings also indicate that combined microdialysis with ESR spectroscopy is a useful in vivo method for monitoring free radical production in the brain.

Animals↗
PubMed1989

Molecular analyses of in vivo hprt mutations in human T-lymphocytes. III. Longitudinal study of hprt gene structural alterations and T-cell clonal origins.

The hprt clonal assay detects mutations occurring in vivo in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of human T-lymphocytes. Analysis of 94 wild-type and 326 hprt mutant clones from 3 normal males was performed using Southern blotting with hprt and T-cell receptor (TCR) gene probes. Gross structural alterations of the hprt gene occurred in approximately 14% of the in vivo derived mutants. Breakpoints were randomly distributed across the gene with one possible mutational "hot spot" observed. Most hprt mutants were independent as judged by TCR gene rearrangement patterns indicating that the measured hprt mutant frequency is a good measure of the actual hprt mutation frequency. However, sibling mutants (generally doublets and triplets except for one nonamer) were detected. Information on the timing in vivo of the hprt mutational events and the persistence in vivo of sibling mutants was also obtained.

Adult↗
PubMed1992

Sequential expression during postnatal development of specific markers of junctional and free sarcoplasmic reticulum in chicken pectoralis muscle.

Skeletal muscle sarcoplasmic reticulum comprises two distinct membrane domains, i.e., the Ca(2+)-pump membrane, corresponding mainly to longitudinal tubules, and the junctional membrane of the terminal cisternae containing the ryanodine receptor/Ca(2+)-release channel. Additional minor proteins previously shown in rabbit fast-twitch skeletal muscle to fractionate selectively to each membrane domain comprise 160- and 53-kDa glycoproteins and 170-kDa low-density lipoprotein (LDL)-binding protein, respectively (Damiani and Margreth, 1991, Biochem. J. 277, 825-832). We report evidence in chicken pectoralis, a predominantly fast muscle, on two closely immunologically related glycoproteins, a minor component of 130-kDa and a major 53-kDa protein. In contrast to the seemingly highly conserved structure of this protein, our results show marked differences in mobilities for chicken 125I-LDL that were detected as a 130- to 116-kDa protein doublet after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, although being otherwise indistinguishable from rabbit 170-kDa protein in LDL-binding characteristics, as well as for preferential association to junctional terminal cisternae. Chicken Ca(2+)-ATPase, although being extensively homologous to rabbit Ca(2+)-ATPase, is shown to be less active and to differ slightly in electrophoretic properties. We have investigated the time course of expression of the specific protein components of longitudinal and of junctional sarcoplasmic reticulum in chick pectoralis muscle from late embryonic development up to 2 months after hatching. Coincident with the posthatching increase in membrane density of high-affinity [3H]ryanodine-binding sites in muscle, both calsequestrin and the species-specific LDL-binding protein(s) are detected in increasing amounts, using ligand blot techniques. In contrast, the appearance and steady accumulation in muscle of Ca(2+)-ATPase, like the time-correlated increase of sarcoplasmic reticulum glycoproteins, are relatively delayed, the most striking changes occurring from 1 week after hatching onward. The sequential expression in chick developing muscle of proteins selectively associated with the junctional terminal cisternae and with longitudinal sarcoplasmic reticulum, respectively, argues for a similar morphogenetic program in avian and mammalian species and, to account for that, for the existence of common epigenetic differentiating influences on the expression of sarcoplasmic reticulum protein genes.

Animals↗
PubMed1989

Embryonic expression of a Drosophila src gene: alternate forms of the protein are expressed in segmental stripes and in the nervous system.

Expression of the Drosophila src-related gene, Dsrc28C has been investigated at the protein level using monoclonal antibodies. This analysis has revealed that the Dsrc28C gene encodes two protein forms: a 66-kD doublet predicted from the sequence of a cDNA clone and an additional 55-kD form. The 66-kD protein doublet is observed first at the cellular blastoderm stage and is not detectable in embryos after 12 hr of development. Expression of the 55-kD protein lags behind that of the 66-kD doublet and then persists throughout embryogenesis. Indirect immunofluorescence microscopy reveals that Dsrc28C protein is localized to the cell periphery during cellular blastoderm and gastrulation. The cell periphery-associated staining is then resolved into ectodermal stripes along the fully extended germ band. After the stripes fade, cytoplasmic staining of the majority of cells within the central and peripheral nervous system is observed. The 66-kD protein was shown to represent the cell periphery-associated form of the protein through antibody staining of larval salivary glands expressing a heat shock promoter-driven, full-length Dsrc28C cDNA. Staining of embryos with a monoclonal antibody specific for the 66-kD protein indicates that the 55-kD protein is the nervous system form. Thus, the 66- and 55-kD proteins are products of the Dsrc28C gene, which exhibit different temporal and spatial patterns of expression in the embryo.

Amino Acid Sequence↗
PubMed1983

Monoclonal antibodies: use to detect developmentally regulated antigens on D. discoideum amebae.

We have used monoclonal antibodies to detect developmentally regulated cell surface antigens on D. discoideum amebae. A study of an antigen detected using an antibody produced by a hybridoma line implicates a previously undescribed component in the process of cell aggregation. This antigen (consisting of a doublet of 69,000 and 73,000 molecular weight) is first detected during the early hours of cell starvation and is present until cells begin slug formation. The developmental appearance of the antigen is not controlled by cAMP pulses and is distinct from that of Contact A sites. Fab fragments directed against the antigen are potent inhibitors of aggregation but do not inhibit the differentiation of cells to aggregation competence.

Adhesiveness↗
PubMed1986

Variation in antigenic determinants specific to the infective stage of Trypanosoma cruzi.

Monoclonal antibodies reactive with the surface antigens of the Peru strain of Trypanosoma cruzi were analyzed by Western blots and immunofluorescence assays to determine their reactivity with three life cycle stages and five strain isolates of T. cruzi. One monoclonal antibody, 7.6, recognized a 68-kilodalton (kDa) polypeptide in Western blots of Peru strain trypomastigotes, epimastigotes, and amastigotes. A 68-kDa polypeptide was also detected by monoclonal antibody 7.6 in trypomastigotes of the CL and Y strains and in the clonal isolates Esmeraldo clone 3 and Silvio X10 clone 1. Positive immunofluorescence results were obtained for all life cycle stages of the five strains that were reacted with monoclonal antibody 7.6, thus indicating that the antigen recognized by monoclonal antibody 7.6 is universally present in all T. cruzi strains tested. In contrast, monoclonal antibody 4.2 reacted with a polypeptide doublet of 90 and 105 kDa in Western blots of Peru strain trypomastigotes, but it did not detect these antigens in epimastigotes or amastigotes. The same polypeptide doublet of 90 and 105 kDa was also detected in Western blots of Y strain trypomastigotes; however, no bands were detected in blots of strain CL or isolate Silvio X10 clone 1 trypomastigotes. In blots of Esmeraldo clone 3 trypomastigotes, a single band of 130 kDa was detected by monoclonal antibody 4.2. In immunofluorescence assays of monoclonal antibody 4.2, positive reactions were obtained only with trypomastigotes of Peru, Y, and Esmeraldo clone 3 strains. Thus, monoclonal antibody 4.2 recognizes a trypomastigote-specific antigen which is not universally present on all strains of T. cruzi.

Animals↗
PubMed2000

c-Fos-related antigens in the central nervous system of an insect, Acheta domesticus.

Fos-related antigens (Fra) were detected in the nuclei of neurones in young adult Acheta domesticus female crickets by immunohistochemical analysis, using an antibody that recognizes the amino-acid sequence 127-152 of c-Fos protein. Specificity of Fra immunoreactivity was confirmed by Western blot analysis of nuclear extracts from neural tissues. A major immunoreactive doublet with an apparent molecular mass of 52,000/54,000 Da was detected in nuclear extracts. Immunostaining of the 52,000/54,000 Da doublet showed variations in intensity during the first 5 days following the imaginal molt. Staining was more intense between day 2 and day 4 when ecdysteroid titers were high. Expression of Fra was low in allatectomized (i.e., deprived of juvenile hormone and ecdysteroids) and ovariectomized (i.e., deprived of ecdysteroids) females as compared to control females. These results show the involvement of hormone-regulated process in expression of Fra. The effect of nociceptive stimulation on Fra expression was tested. Twenty minutes after removal of the ovipositor, a supplementary band with an apparent molecular mass of 70,000 Da appeared in the nuclear extracts, then decreased and disappeared totally after 45 min. Several other Fos-related antigens with different temporal patterns of expression were also detected.

Animals↗
PubMed1991

Detection and characterization of leptospiral antigens using a biotin/avidin double-antibody sandwich enzyme-linked immunosorbent assay and immunoblot.

A biotin/avidin double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of antigens of Leptospira interrogans serovars in experimentally inoculated bovine urine samples was evaluated. Immunoglobulin G (IgG) from rabbits immunized with L. interrogans serovar hardjo type hardjobovis sonicated, whole cell, and formalinized-heated antigen preparations were purified by a protein A-superose column coupled to fast protein liquid chromatography, and evaluated for species specificity in the ELISA. The ELISA using each specific IgG detected as few as 10(4) leptospires of the homologous serovar hardjo diluted in phosphate-buffered saline solution with Tween 20 (PBSS-Tween 20). On immunoblot analysis of proteinase-K-digested whole cell leptospiral preparations, each IgG revealed the presence of bands specific to serovar hardjo, suggesting the presence of serovar-specific epitopes on the lipopolysaccharide molecules. The minimum number of cells of heterologous serovars pomona, grippotyphosa, bratislava, icterohaemorrhagiae and copenhageni detected by each ELISA was greater, ranging from 10(6) to 10(7). The common antigenic determinants observed on immunoblot analysis were different for each specific IgG, except for a major cross-reacting, possibly flagellar, protein doublet at approximately 36-36.5 kDa. Leptospires were equally well detected by the ELISA in both bovine urine and PBSS-Tween 20.

Animals↗