Application of monoclonal antibody, specific for intracellular Orientia tsutsugamushi, to immunofluorescent antibody test for determining antibiotic susceptibility

The simple quantification of viable intracellular bacteria is important for the study of an obligate intracellular bacterium, Orientia tsutsugamushi. We applied a novel monoclonal antibody (M686-13)--specific for intracellular Orientia--to an immunofluorescent antibody (IFA) test for determining antibiotic susceptibility of O. tsutsugamushi. M686-13 did not react with Orientia that was inhibited by doxycycline, although bacterial particles still remained in the cells. This preferential staining of proliferating bacteria made the IFA test rapid and precise. Using this method, we could successfully measure the minimal inhibitory concentration (MIC) of a Korean strain of O. tsutsugamushi to doxycycline and clindamycin. This method may be used in other procedures to evaluate the growth of Orientia.     

Novel polysaccharide antigen of Orientia tsutsugamushi revealed by a monoclonal antibody

Orientia tsutsugamushi , the causative agent of scrub typhus, is an obligate intracellular bacterium that replicates in the cytosol of host cells. Although several protein antigens have been characterized and cloned, little information exists regarding the polysaccharide antigen of this bacterium. In this study, we identified and characterized a novel antigen defined by a monoclonal antibody (MAb), NT19, against O. tsutsugamushi . Immunofluorescence microscopic studies showed that the NT19 antigen is released from the bacteria in the cytosol of host cells forming aggregates with bacteria. Immunoblot analysis showed that MAb NT19 recognized a strong band with a molecular mass of 20 kDa that was resistant to proteinase K digestion and sensitive to periodate oxidation, suggesting that the NT19 antigen is a polysaccharide. The function of this polysaccharide is not known, but considering its distribution within a bacterial microcolony, it is suspected to be involved in forming a biofilm-like structure within host cells.     

Plasmodium falciparum: an invasion inhibitory human monoclonal antibody is directed against a malarial glycolipid antigen

A Plasmodium falciparum malaria blood stage antigen was detected using a human monoclonal antibody (MAb A52A6) obtained from a clinically immune donor. Immunofluorescence analysis showed that the MAb reacted with the intracellular parasite throughout the asexual blood stage cycle as well as with gametocytes. The MAb also reacted with the surface of erythrocytes containing late stage P. falciparum parasites. The antigen seen by the MAb was species- but not strain- or isolate-specific. At rupture of the infected erythrocytes, antigenic material was deposited on the membrane of uninfected cells surrounding the parasite. At merozoite invasion MAb reactive material was present on the invaginating erythrocyte membrane, indicating an involvement of the antigen in the invasion process. This was also indicated by the high capacity of the MAb to inhibit merozoite invasion in vitro. The antigen appears to be a phosphoglycolipid, sensitive to phospholipase and present in lipid extracts of P. falciparum-infected erythrocytes.     

Malignant ascites of serous papillary ovarian adenocarcinoma. An immunocytochemical study of the tumor cells

In 17 malignant peritoneal effusions due to papillary serous adenocarcinoma of the ovary, the reaction patterns of the tumor cells to monoclonal antibodies (MAbs) against surface antigens were studied and compared with the reaction patterns of mesothelial cells in the same effusions. The following surface markers were used with the adhesive slide method: epithelial membrane antigen (EMA), human epithelium-specific cell surface antigen (HEA-125), human endothelial antigen (BMA-120), carcinoembryonic antigen (CEA 3-13), an antibody against natural killer cells and cytotoxic cells (BMA-070), granulocyte antigen (Leu M1) and leukocyte antigen of class I (HLA-1). In all cases, from 30% to 95% of the tumor cells reacted with EMA and HEA-125. Tumor cells showed a positive staining with CEA 3-13 in only five cases. In all cases, from 75% to 95% of the tumor cells reacted positively with BMA-120. The reactivity of a few mesothelial cells with EMA and of all mesothelial cells with BMA-120 did not interfere with the identification of positive tumor cells since the reaction patterns were different. Interestingly, our study demonstrated that BMA-070, an MAb identifying natural killer cells and cytotoxic cells, is also a most useful tumor marker. The same was found to be true for Leu M1, an MAb originally thought to react only with granulocytes. The tumor cells showed a partial or total loss of the expression of HLA-1 reactivity. Since all cases were immunocytochemically positive for tumor cells while conventional cytology was positive in only 13 of the cases, the immunocytochemical analysis of malignant peritoneal effusions due to papillary serous adenocarcinoma of the ovary seems able to improve the cytologic diagnosis of the fluids.     

Role of Myxococcus xanthus cell surface antigen 1604 in development.

The inhibition of development of Myxococcus xanthus by monoclonal antibody (MAb) 1604 has been further investigated with two MAbs produced against the affinity-purified cell surface antigen (CSA) 1604. Both of these second-generation MAbs, 4070 and 4054, reacted with the same band at 150 kilodaltons (kDa) on Western immunoblots of lysed and reduced cells. This band was also identified by MAb 1604. However, the affinity-purified CSA was a complex of the two proteins (51 and 23 kDa) and lipopolysaccharide (LPS) that the 150-kDa material comprised. One of the three MAbs, 4070, reacted with LPS on Western immunoblots. Another MAb, 4054, reacted with the 23-kDa protein, and MAb 1604 reacted with the 51-kDa protein found in the CSA complex. Competitive binding studies verified that MAbs 4054 and 1604 identified different epitopes, and MAb 4070 probably reacted with a third epitope of the CSA 1604 complex. MAb 4054 blocked development, although not as thoroughly as MAb 1604 did, when added at 60 micrograms/ml to cells undergoing submerged development. In contrast, MAb 4070 prevented sporulation in submerged development and induced the cells to reaggregate in rings around the initial aggregation centers. A mutant strain of M. xanthus that is deficient in the epitope for MAb 1604 retained the epitope for MAb 4054. The affinity-purified antigen 1604, when added to cells at greater than or equal to 550 ng/ml, altered the appearance of the fruiting bodies and at higher concentrations prevented fruiting body formation. The CSA 1604 moiety responsible for this inhibitory effect is apparently a peptide constituent and not the LPS.     

Matrix protein-specific IgA antibody inhibits measles virus replication by intracellular neutralization

Measles virus (MV) is still an imposing threat to public health. The matrix (M) protein has been shown not only to function as a structure block in the assembled MV virions, but also to regulate viral RNA synthesis, playing an important role in MV's replication and assembly. In the present study, we generated a panel of IgG monoclonal antibodies (MAbs) against M protein and successfully obtained one IgA MAb (5H7) from the IgG panel. Employing the polarized Vero cells grown in the two-chamber transwell model, we investigated whether M-specific 5H7 IgA MAb could suppress MV's replication and assembly. The data presented indicate that, while failing to show the activities of traditional neutralization and immune exclusion, M-specific IgA MAb was able to effectively inhibit viral replication by intracellular neutralization (78%), supporting the notion that the M protein is important for MV assembly and replication and implying that the M protein was an effective target antigen. The data also showed that MV had a long entry and assembly phase during viral replication, providing an extended window for IgA intervention. The colocalization of M proteins and M-specific 5H7 IgA MAbs demonstrated that the intracellular neutralization was due to the direct binding of the M-specific 5H7 IgA MAbs to the M proteins. In summary, the present study has added another example showing that IgA antibodies targeting internal viral antigens could proactively participate in mucosal immune protection by intracellular neutralization and has provided evidence that M protein might be included as a target antigen in future MV vaccine design.     

Identification of a novel T cell surface disulfide-bonded dimer distinct from the alpha/beta antigen receptor

Hybridomas were prepared from the spleen of a BALB/c mouse immunized with EL-4 T lymphoma cells. One, designated A1, was found to secrete a monoclonal antibody that reacted with two T lymphoma cells of C57BL origin, EL-4 and C6VLB, but not with normal C57BL/6 splenocytes or thymocytes, C57BL/6 T cell clones, or other T or B lymphomas by complement-mediated cytotoxicity or indirect immunofluorescent staining. Monoclonal antibody (MAb) A1 precipitated a protein that migrated at 85 kD under nonreducing and 43 kD under reducing conditions. The fact that the antigen defined by MAb A1 was a disulfide-linked dimer, together with the essentially clone-specific distribution of the reactive epitope, raised the possibility that the antibody defined an epitope of the antigen receptor. However, several additional observations revealed that the antibody defined a distinct and novel T cell surface structure. MAb 124-40, previously shown to react with the antigen receptor of C6VLB cells, reacted with variants of C6VLB that failed to express the A1 epitope. Sequential immunoprecipitation indicated that MAb A1 and MAb 124-40 reacted with distinct molecular species on C6VLB cells. Endoglycosidase digestion showed that the structure reactive with MAb A1 was not derived from that reactive with MAb 124-40 by addition of N-linked oligosaccharide residues. Two-dimensional gel electrophoretic analysis of precipitates obtained from radioiodinated C6VLB cells with MAb 124-40 resolved the alpha and beta subunits of the antigen receptor. Similar analysis of precipitates obtained with MAb A1 revealed only a single basic chain under reducing conditions, although anomalous mobility suggestive of a second, more acidic chain was observed under nonreducing conditions. Two-dimensional maps of tyrosine-containing chymotryptic peptides of the proteins isolated with MAb A1 and MAb 124-40 were completely different, suggesting that the molecules shared no peptides and were distinct in primary structure. Finally, cross-linking studies performed with a cleavable reagent indicated that the A1 molecule, unlike the antigen receptor defined with MAb 124-40, was not associated with additional, T3-like structures on the surface of C6VLB cells. Although the MAb A1 was unreactive with normal cells in cytotoxicity or staining assays, a molecule of the appropriate size was immunoprecipitated in small amounts from lysates of radioiodinated normal spleen and thymus cells. These data indicate that MAb A1 defines a novel disulfide-linked T cell surface molecule distinct from the antigen receptor.     

Ultrastructural localization of epitopes recognized by monoclonal antibodies produced to rat Pneumocystis carinii.

The binding sites of five monoclonal antibodies (MAb) developed against rat Pneumocystis carinii were examined at the ultrastructural level by using a post-embedding labeling method. Although all five MAb reacted with the pellicle of P. carinii, they were divided into two groups by localization of binding sites. The MAb 168.2.1, 174.2.1, and 215.2.1 reacted mainly with the electron-dense outer layer, whereas MAb 227.1.1 and 228.1.1 labeled both the outer dense layer and the middle lucent layer. With in the first group of MAb, no significant differences were observed in the reactivity patterns seen with the different stages of P. carinii. In the second group, however, the intensity of labeling of the electron-dense layer was higher in the precyst, cyst, and ruptured cyst stages than in the trophozoite stage. These latter results indicate that there may be an increase in antigen accumulation during development from the trophozoite to the cyst stages, or that antigens may be modified the development.     

Differential localization within human kidney of five membrane proteins expressed on acute lymphoblastic leukemia cells

The reactivity of a panel of monoclonal antibodies (MAb) produced against non-T, non-B acute lymphoblastic leukemia cells was investigated by immunoperoxidase staining of sections of normal human kidney. The antigens of kidney reactive with the MAb were isolated by immunoaffinity chromatography and were purified further by immunoprecipitation. Two MAb, 44D7 and 44H9, reacted with determinants found exclusively on the basolateral membranes of proximal convoluted tubules. The 44D7 antigen isolated from kidney was biochemically similar to that isolated from leukemic cells. It was resolved as a multimeric complex with an apparent m.w. of 120,000 when analyzed by SDS-PAGE under nonreducing conditions. The 44H9 antigen has not yet been purified from kidney. MAb 50B4 reacted with components of the interstitium and with the mesangium of glomeruli. It immunoprecipitated a polypeptide chain of apparent m.w. 85,000, similar to that of the 50B4 antigen isolated from leukemic cells. MAb 44G4 also reacted with the mesangium of glomeruli and with the interstitium of the kidney. However, the endothelium of glomerular capillaries and of interstitial blood vessels has also reacted with MAb 44G4. The kidney antigen recognized by MAb 44G4 was characterized as a major polypeptide band, 95,000 m.w. (reduced) and 125,000 m.w. (nonreduced), a subunit structure analogous to the 44G4 antigen isolated from leukemic cells. MAb 44E3 reacted with all cellular elements of glomeruli, tubules, blood vessels, and interstitium. Two polypeptide chains of apparent m.w. 94,000 and 90,000 were immunoprecipitated from kidney by MAb 44E3, while a single polypeptide chain of 94,000 m.w. was precipitated from leukemic cells. Our results describe five new antigens with distinctive cellular distributions within kidney.     

Monoclonal antibody specific for Listeria monocytogenes associated with a 66-kilodalton cell surface antigen.

A monoclonal antibody (MAb), EM-7G1, specific for Listeria monocytogenes was developed by using a previously developed MAb, C11E9 (A. K. Bhunia, P. H. Ball, A. T. Fuad, B. W. Kurz, J. W. Emerson, and M. G. Johnson, Infect. Immun. 59:3176-3184, 1991), to mask epitopes shared by L. monocytogenes and Listeria innocua in a 66-kDa cell surface protein. MAb EM-7G1 was an immunoglobulin subclass G1 antibody with kappa light chains. This MAb reacted with all 34 strains of L. monocytogenes tested and showed no cross-reaction with other Listeria spp. or other gram-positive or gram-negative organisms tested by enzyme-linked immunosorbent assay, dot blotting, and colony blotting. A second MAb, EM-6E11, reacted with all Listeria spp. tested but no other bacteria. In a Western blot (immunoblot) assay, EM-7G1 reacted with a crude cell surface protein of 66 kDa with a pI value of 6.7, while EM-6E11 reacted with two protein bands of 43 and 94 to 97 kDa with pI values of 4.0 and 4.3, respectively. Results with trypsin or pronase treatments indicated that the cell antigen reacting with EM-7G1 was on the surface of L. monocytogenes V7 and Scott A cells.     

Immunofluorescence of the epizootic ulcerative syndrome pathogen,Aphanomyces invadans,using a monoclonal antibody

A monoclonal antibody (MAb), designated 3gJC9, was raised against a protein antigen of Aphanomyces invadans, the oomycete pathogen that causes epizootic ulcerative syndrome (EUS). The antigen was expressed on the surface of hyphae and secreted extracellularly. MAb 3gJC9 did not cross-react with other oomycete or fungal pathogens of fish, although it did react to the crayfish plague pathogen A. astaci. The MAb was used for immunofluorescent staining on histological sections of fish infected with EUS, and was found to be more sensitive than conventional staining methods for detecting A. invadans. It thus has utility in confirming the case definition of EUS. It also revealed very small filamentous structures, the significance of which is unclear, but they may represent an early stage of infection, thus allowing earlier detection of the disease, since they are not detected using conventional staining methods.     

Expression of blood group A and B antigens in nuclear heterochromatin in mucous cells of human cervical glands.

Using a post-embedding immunogold labeling procedure, we found that monoclonal antibody against A (MAb-A) or B antigen (MAb-B) reacted with nuclear heterochromatin regions, as well as secretory granules, in mucous cells of human cervical glands. Systematic and critical observation of specimens from 24 individuals of different blood groups revealed that the labeling pattern with MAb with strictly dependent on the blood group (A,B, or O) of the donors, i.e., MAb-A reacted with the heterochromatin from blood group A and AB but not with B and O individuals. Labeling with MAb-B was also specific for the heterochromatin from blood group B donors. On the other hand, MAb against H antigen did not react with the heterochromatin from any individuals examined, despite the fact that H antigens were detected by the MAb in secretory granules. Such specific reactions provide evidence that certain types of blood group-related antigens exist in the nuclear heterochromatin in mucous cells of human cervical glands. In contrast to the secretory granules in which ABH antigens were recognized by blood group-specific lectin, heterochromatin regions had little or no affinity for these lectins. Furthermore, the secretory status of individuals affected the staining intensity with MAb in secretory granules but not in the heterochromatin. These results suggest that the blood group substances found in the heterochromatin may have different molecular properties from those in the secretory granules, although both have the same determinant structures of ABH antigens.     

Effects of maturational agents on expression and secretion of two partially characterized high molecular weight milk-related glycoproteins in MCF-7 breast carcinoma cells

We have previously defined a human mammary epithelial antigen using a murine monoclonal antibody (MAb), designated DF3, prepared against a membrane-enriched fraction of a human breast carcinoma. MAb DF3 detects a cell surface antigen with a molecular weight (mw) of approximately 300 Kd and a higher mw species also detectable in human milk. These findings and the demonstration that butyric acid (BA) increases DF3 antigen expression suggested that MAb DF3 reacts with a differentiation antigen detectable in human breast carcinoma cells. The results of the present study demonstrate that MAb DF3 reacts with two mucin-like high mw glycoproteins (330 and 450 Kd) present in MCF-7 breast carcinoma cells. The results also demonstrate that the intracellular content and secretion of DF3 antigen is increased by 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-beta-D-arabinofuranosylcytosine (ara-C). Other known inducers of differentiation including retinoic acid (RA), hexamethylene bisacetamide (HMBA), 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) and certain polar solvents decrease DF3 antigen expression. Furthermore, the results demonstrate that DF3 antigen is secreted and that the extent coincides with changes in intracellular content. Finally, actinomycin D and cycloheximide inhibit the increases in DF3 antigen expression following TPA treatment thus suggesting that newly synthesized RNA and protein are required for induction of this antigen. Thus, the monitoring of DF3 antigen expression may provide a marker for studying maturation of human breast cancer cells.     

Exposed epitopes on a Trypanosoma equiperdum variant surface glycoprotein altered by point mutations.

African trypanosomes are covered by a dense protein layer that is immunologically distinct on different trypanosome isolates and is termed the variant surface glycoprotein (VSG). The different VSGs are expressed in a general order, where some VSGs appear preferentially early in infection and others only later. The exposed epitopes on a late antigen, VSG 78, of T.equiperdum were studied by the technique of monoclonal antibody (MAb) escape selection. MAbs that neutralize trypanosomes bearing VSG 78 reacted with the VSG only when it was attached to the trypanosome surface, suggesting that the most immunogenic surface epitopes are conformational. Trypanosome clones resistant to one of the MAbs yet still expressing VSG 78 or 78(20) were isolated in vitro. Two independent variants resistant to MAb H3 changed Ser192 to Arg by a single base change in the VSG gene and a variant resistant to MAb H21 had a single base change that converted Gln172 to Glu. A variant resistant to MAb H7 had several changes in the VSG gene, a gene conversion in the 5' region and an isolated mutation in codon 220 that is proposed to be responsible for the resistance phenotype. The isotypic bias of the MAbs against VSG 78 and an analysis of the natural variants that are resistant to MAb 78H21 suggest that glycosylation plays a role in the immunogenicity of these proteins. The analysis defines some of the exposed amino acid residues and demonstrates that VSG genes are altered by mutations and small gene conversions as well as replaced by large gene conversion-like events. The results provide biological data supporting the model of VSG structure obtained by crystallographic studies.     

Purification and properties of Myxococcus xanthus cell surface antigen 1604.

A cell surface antigen complex from Zwittergent-solubilized Myxococcus xanthus has been purified by immunoaffinity chromatography with monoclonal antibody (MAb) 1604 and by subsequent gel filtration. We propose that the cell surface antigen (CSA) 1604 complex participates in intercellular interactions. The apparent total molecular mass of the CSA 1604 complex is 200 kilodaltons (kDa), as determined by gel filtration and by electrophoresis and Western immunoblot probing with MAb 1604. The antigen epitope recognized by MAb 1604 is on a 51-kDa polypeptide. The CSA complex also contains 14% neutral carbohydrate and a 23-kDa polypeptide that lacks the 1604 epitope. The carbohydrate is most likely part of a lipopolysaccharide (LPS) associated with the CSA, because an MAb recognizing an O antigen epitope from the LPS of M. xanthus also reacted with CSA 1604 on Western immunoblots. Thus, the 200-kDa CSA complex consists of 97 +/- 6 kDa of protein and many associated LPS molecules. The LPS evidently produces the multiplicity of bands observed on Western immunoblots between 100 and 200 kDa. The association with LPS may contribute to the negative charge of the CSA 1604 complex, which has a pI of 4.3. The CSA was clustered on the surface of intact M. xanthus cells after labeling with MAb 1604 and immunogold. Furthermore, fractionation studies indicated that cells grown on a plastic surface had 50% of their total CSA 1604 in the cytosol, 39% in the membrane fraction, and 8% in the periplasm. Saturable binding studies with 125I-MAb 1604 indicated that there were 2,400 CSA 1604 sites per cell. The Kd for MAb 1604 binding to the cell was 9 nM.     

Enhanced susceptibility of multidrug resistant strains of Mycobacterium tuberculosis to granulysin peptides correlates with a reduced fitness phenotype

Previously it was shown that the antimicrobial protein granulysin possesses potent membranolytic activity against Mycobacterium tuberculosis. Here we demonstrate that granF2 and G13, which are two short synthetic peptides derived from granulysin, inhibited the in vitro growth of clinical isolates of both multidrug resistant and drug susceptible strains of M. tuberculosis. Importantly, a particularly high activity against multidrug resistant M. tuberculosis correlated with a reduced growth rate compared to drug susceptible strains. A synergistic antibacterial effect of granF2 was further observed in combination with ethambutol, a compound with a documented effect on cell wall permeability. This finding suggests that granF2 and ethambutol exert their functions at different levels of the mycobacterial surface. Upon infection of macrophages in vitro, granF2 but not G13 efficiently reduced the intracellular growth of multidrug resistant M. tuberculosis in the presence of the pore-forming protein streptolysin O. The apoptotic function of granF2 apparently promoted destruction of host cells whereby the peptide gained access to and killed intracellular bacteria. Thus, a cost of resistance and a subsequent reduced fitness, measured as decreased growth among multidrug resistant strains of M. tuberculosis, could be associated with increased susceptibility to natural immune defense mechanisms, such as antimicrobial peptides of granulysin. However, a robust cell wall and the membrane of cells still provide physical shelter for the bacteria that may spare sensitive M. tuberculosis stains from being killed.     

Cell cycle kinetics and monoclonal antibody productivity of hybridoma cells during perfusion culture

The flow-cytometric (FCM) analysis of bivariate DNA/lgG distributions has been conducted to study the cell cycle kinetics and monoclonal antibody (MAb) production during perfusion culture of hybridoma cells. Three different perfusion rates were employed to demonstrate the dependency of MAb synthesis and secretion on cell cycle and growth rate. The results showed that, during the rapid growth period of perfusion culture, the level of intracellular igG contents of hybridoma cells changed significantly at each perfusion rate, while the DNA histograms showing cell cycle phases were almost constant. Meanwhile, during the reduced growth period of perfusion culture, the fraction of cells in the S phase decreased, and the fraction cells in the G1/G0 phase increased with decreasing growth rate. The fraction of cells in the G2/M phase was relatively constant during the whole period of perfusion culture. Positive correlation was found between mean intracellular IgG contents and the specific MAb production rate, suggesting that the deletion of intracellular IgG contents by a flow cytometer could be used as a good indicator for the prediction of changes in specific MAb productivity following manipulation of the culture condition. (c) 1994 John Wiley & Sons, Inc.     

Different modes of sialyl-Tn expression during malignant transformation of human colonic mucosa

Monoclonal antibodies TKH2 and B72.3, which react with the mucin-associated sialyl-Tn(STn) antigen, preferentially bind to cancerous but not normal colonic tissues. If O-acetyl groups are removed by saponification of tissues, MAb TKH2 will react with normal colonocytes, whereas MAb B72.3 remains non-reactive. To explain this difference in binding specificity, we tested both MAbs against synthetic constructs of single (monomeric) or clustered (trimeric) STn epitopes by enzyme immunoassay. Both MAb TKH2 and MAb B72.3 reacted with trimeric STn, but MAb TKH2 demonstrated greater binding than MAb B72.3 to monomeric STn. This suggests that normal colonic mucosa expresses monomeric STn epitopes, but that with transformation to malignancy, clustered STn epitopes appear. The appearance of clustered STn epitopes during colonic carcinogenesis represents a novel pattern of carbohydrate antigen expression and implicates alterations at the level of apomucins and/or glycosyltransferases responsible for cluster epitope formation.     

A monoclonal antibody to chicken gizzard desmin that recognizes intermediate filaments and nuclear granules in BHK21/C13 cells

One hybridoma (AC54), which produces monoclonal antibody (MAb) that recognizes both intermediate filaments (IFs) and nuclear granules in BHK21/C13 cells, and two hybridomas (AC19 and AC36) which produce MAbs that recognize IFs only, were obtained by using a crude actin preparation from chicken gizzard as an antigen. In immunoblotting, both the AC54 and AC19 MAbs reacted with the 52 kD protein (desmin) and some other proteins in gizzard and BHK21/C13 cells. Indirect immunofluorescent microscopy of BHK21/C13 cells showed that the cytoplasmic filaments stained by these MAbs were IFs based on their colchicine-induced whorl formation. The ability of AC54 MAb to recognize IFs was more limited than that of AC19 MAb. The nuclear granules recognized by AC54 MAb were in a different location than the cytoplasmic IFs and sometimes were concentrated in the nucleolus. These results indicate that AC54 MAb is an anti-desmin MAb that reacts with some desmin-related proteins; that it recognizes IFs differently than AC19 MAb, another anti-desmin MAb; and that it recognizes nuclear granules in locations where desmin or desmin-related protein has not yet been reported.     

Production of monoclonal antibodies against a novel glycoprotein synthesized and secreted by dog thyroid C-cells.

A monoclonal antibody (MAb) that reacted only with thyroid C-cells was raised against cell suspensions from dog thyroid glands, to examine a glycoprotein secreted by C-cells. After chronically-induced hypercalcemia and administration of an anti-thyroid drug, reaction products for the antibody markedly decreased in C-cells, coinciding with alterations in calcitonin immunoreactivity. The antigen recognized by the MAb appears to be a secretory protein. The MAb reacted with C-cells from a wide variety of mammalian species, including rats, mice, hamsters, cattle, cats, rabbits, and monkeys. Furthermore, tumor cells of human medullary thyroid carcinoma, which is derived from C-cells, were immunoreactive to the MAb. Exceptionally, C-cells from guinea pigs and pigs were not stained with the MAb. No crossreactivity was observed in any of the dog tissues examined. Immunoblot analysis demonstrated that the MAb recognized a single prominent band at a molecular weight of approximately 79,000. The 79 KD band reacted with various digoxigenin-labeled lectins, including GNA, DSA, SNA, and MAA; it is a glycoprotein containing mannose, N-acetylglucosamine, and sialic acid. Dog thyroid C-cells were also densely stained with these lectins. The results indicate that thyroid C-cells synthesize and secrete a specific glycoprotein in addition to peptide hormones.