Production and characterisation of monoclonal antibodies to salmon pancreas disease virus.

Six mouse monoclonal antibodies (mAbs) specific to salmon pancreas disease virus (SPDV) were produced following immunisation with purified virus preparations. These mAbs and 2 mAbs resulting from an earlier investigation were characterised. None of the mAbs possessed virus neutralising activity but all reacted with 4 geographically different SPDV isolates as determined by indirect immunofluorescence. Three mAbs produced positive immunostaining with Western blots of SPDV proteins. The 4H1 mAb reacted with the 53 kDa structural E1 glycoprotein present in virus-infected cells and in gradient-purified virus. Two mAbs, 5A5 and 7B2, which exhibited unusual immunofluorescence staining of the nuclear margin, reacted with a 35 kDa protein, which is present in gradient-purified virus and which is considered to be the capsid protein. A sandwich ELISA, based on the use of mAb 2D9 for capture and a biotinylated conjugate of mAb 7A2 for detection, detected SPDV antigen in virus-infected Chinook salmon embryo-214 cells and gradient-purified virus. These mAbs may be of use in pathogenesis studies and in diagnostic test development.     

Reactivity of anti-proliferating cell nuclear antigen (PCNA) murine monoclonal antibodies and human autoantibodies to the PCNA multiprotein complexes involved in cell proliferation

Proliferating cell nuclear Ag (PCNA) occurs as a component of multiprotein complexes during cell proliferation. We found the complexes to react with murine anti-PCNA mAbs, but not with anti-PCNA Abs in lupus sera. The complexes were purified from rabbit thymus extract by affinity chromatography using anti-PCNA mAbs (TOB7, TO17, and TO30) and analyzed by ELISA, immunoprecipitation, immunoblotting, and HPLC gel filtration. That PCNA was complexed with other proteins was demonstrated by its copurification with a group of proteins excluded by an HPLC G3000 SW column. Although immunoblot analysis showed the mAbs to react exclusively with the 34-kDa PCNA polypeptide, they nonetheless immunoprecipitated the same group of proteins, confirming the interaction of the isolated PCNA with other proteins. Anti-PCNA sera, including AK, which reacts with biologically functional sites on PCNA, did not react with complexed PCNA, but did react with it once it was dissociated from the complexes. PCNA complexes in turn reacted with murine anti-DNA mAbs, as well as with Abs against p21, replication protein A, DNA helicase II, cyclin-dependent kinases 4 and 5, and topoisomerase I. These findings suggest that the PCNA complexes purified using anti-PCNA mAbs comprise the "protein machinery" for DNA replication and cell cycle regulation. They also suggest that anti-PCNA mAbs are useful tools with which to characterize the protein-protein interactions within PCNA complexes, as well as the autoimmune responses to proteins interacting with PCNA, which may shed light on the mechanisms of autoantibody production in lupus patients.     

Identification of salivary proteins in Aedes togoi (Theobald) that bind to mouse immunoglobulin E

Mosquito saliva contains a number of immunogenic molecules that can induce allergic reactions in host animals. The oriental tiger mosquito, Aedes togoi (Theobald), feeds on both humans and animals and is also a vector of various pathogenic viruses. However, information on immunogenic antigens in the saliva of this mosquito species is scarce. In this study, we used immunohistochemical staining and confocal microscopy to determine the fine structure of the acini lobes of salivary glands of A. togoi. In addition, we identified proteins from salivary gland extracts that reacted to mouse immunoglobulin E in an immunoblot analysis. At least nine proteins, ranging in size from 10 to 145 kDa, bound to IgEs; a 33 and 57.5 kDa protein showed the strongest binding signal. Our results provide important information on the identities of potential allergens in mosquito saliva.     

Development of monoclonal antibodies reacting against mycobacterial 65 kDa heat shock protein by using recombinant truncated products.

A mycobacterial 65 kDa molecule is a member of the GroEL heat shock protein family. We developed mAbs reacting against recombinant 65 kDa protein by using a gene (pTB12) which encodes this protein. Three mAbs (B20, B97 and B167) reacted selectively with 65 kDa proteins of Mycobacterium tuberculosis, BCG and Mycobacterium leprae, although B20 and B167 may weakly react with a 15 kDa molecule of mammalian cells. One (B108) was obviously cross-reactive between mycobacterial 65 kDa and the mammalian intracytoplasmic protein. We also developed deletion mutants of pTB12. The localization of these mAb-defined epitopes was determined by using truncated proteins of the Mycobacterium tuberculosis 65 kDa molecule produced in E. coli. Immunohistochemical analysis showed that B20, B97 and B167 mAbs could detect this antigen in experimental granulomas induced by injection of BCG in the subcutaneous tissue of rats. These mAbs should be useful for analyzing the immunobiologic roles of mycobacterial 65 kDa molecules.     

Glutathione-binding proteins identified by monoclonal antibodies which depress the behavioral response evoked by glutathione in Hydra.

Hydra shows at least 5 components of the behavioral response (R1-R5) which are evoked at different concentrations of S-methylglutathione (GSM). We have prepared several monoclonal antibodies (mAbs), each of which depressed specific responses and visualized specific structures. In this paper, we analyzed molecules participating in the signaling pathway of R5 response with use of three mAbs (J245, J5 and J5/1), all of which depressed the response. A behavioral analysis of the response in the presence of mixtures of these mAbs suggested that J245 and J5 both acted on a component of the receptor-effector system which was not affected by J5/1. Correspondingly, an immunoblotting analysis showed two 220 kDa proteins which reacted with both J245 and J5 but not with J5/1. ELISA analyses also showed that the J245 antigens formed only a portion of the J5 antigens. A labeled major peak was found in the 220 kDa protein fraction by gel permeation HPLC after immunoprecipitation of the J245 antigen photolabeled with [35S]-S-(p-azidophenacyl)-glutathione. Competition of the photolabeling by the ligands GSM and L-glutamate (a competitive inhibitor of the R5 response) indicated dissociation constants for their binding to the protein of 55 and 90 microM, respectively. These values were consistent with those expected from behavioral experiments. The 220 kDa proteins therefore appear to be candidates for the receptor molecules mediating R5.     

Characterization of mosquito larvicidal parasporal inclusions of a Bacillus thuringiensis serovar higo strain

The parasporal inclusion proteins of the type strain of Bacillus thuringiensis serovar higo (H44), that have moderate mosquitocidal activity, were characterized. The purified parasporal inclusions, spherical in shape, were examined for activity against the two mosquito species, Culex pipiens molestus and Anopheles stephensi and the moth-fly, Telmatoscopus albipunctatus . The LC₅₀ values of the inclusion for the two mosquitoes were 3·41 and 0·15 μg ml⁻¹, respectively. No mortality was shown for T. albipunctatus larvae by the inclusions at concentrations up to 1 mg ml⁻¹. Solubilized parasporal inclusions exhibited no haemolytic activity against sheep erythrocytes. Parasporal inclusions consisted of eight proteins with molecular masses of 98, 91, 71, 63, 59, 50, 44 and 27 kDa. Of these, the 50 and 44 kDa proteins were the major components. Analysis with immunoblotting revealed that, among several inclusion proteins of B. thuringiensis serovar israelensis, only two proteins of 130 kDa and 110 kDa reacted weakly with antibodies against higo proteins. N-terminal amino acid sequences of the 98, 91, and 71 kDa proteins showed 85–100% identity to those of the two established Cry protein classes, Cry4A and Cry10A.     

Conversion of Allethrolone to cis-Jasmone

The potential allergenic proteins in beef were investigated. The sera of ten beef-allergic patients suffering from atopic dermatitis and having a positive RAST score to beef, aged 3-18 years, were obtained from Yoshida Hospital in Japan, and five non-allergic individuals were subjected to this study. The sera of the ten patients reacted strongly to a beef extract, but not to pork and chicken extracts by both ELISA and immunoblotting. The sera of the five control subjects did not react to any of these meat extracts. Three bands having molecular masses of ~200 kDa, ~67 kDa and ~60 kDa were observed by immunoblotting after SDS-PAGE. Two fractions of the beef extract from a Sephadex-gel (G-200) filtration column strongly reacted with the sera of the beef-allergic patients by ELISA and immunoblotting: one fraction had the ~67 kDa component and the other had the ~200 kDa and ~60 kDa components. One of them (~67 kDa) was confirmed to be bovine serum albumin (BSA) by an analysis of the N-terminal amino acid sequence. We could not identify the others by sequencing, but the ~200 kDa and ~60 kDa components were presumed to be glycoproteins. Bovine gamma globulin (BGG: M.W. ~160 kDa) is a glycoprotein and has several subunits. The beef-allergic patients showed strong reactivity to the ~200 kDa and ~60 kDa components of pure BGG by immunoblotting. Inhibition-ELISA showed that pure BGG preparations strongly inhibited the binding of sera from the beef-allergic patients to the beef extract. These results suggest that the ~200 kDa, ~67 kDa and ~60 kDa components in the beef extract had strong allergenicity: ~67 kDa was BSA, and ~200 kDa and ~60 kDa were presumably aggregated BGG and it’s heavy chain, respectively.     

Meat allergy: investigation of potential allergenic proteins in beef

The potential allergenic proteins in beef were investigated. The sera of ten beef-allergic patients suffering from atopic dermatitis and having a positive RAST score to beef, aged 3-18 years, were obtained from Yoshida Hospital in Japan, and five non-allergic individuals were subjected to this study. The sera of the ten patients reacted strongly to a beef extract, but not to pork and chicken extracts by both ELISA and immunoblotting. The sera of the five control subjects did not react to any of these meat extracts. Three bands having molecular masses of approximately 200 kDa, approximately 67 kDa and approximately 60 kDa were observed by immunoblotting after SDS-PAGE. Two fractions of the beef extract from a Sephadex-gel (G-200) filtration column strongly reacted with the sera of the beef-allergic patients by ELISA and immunoblotting: one fraction had the approximately 67 kDa component and the other had the approximately 200 kDa and approximately 60 kDa components. One of them (approximately 67 kDa) was confirmed to be bovine serum albumin (BSA) by an analysis of the N-terminal amino acid sequence. We could not identify the others by sequencing, but the approximately 200 kDa and approximately 60 kDa components were presumed to be glycoproteins. Bovine gamma (BGG:globulin M.W. approximately 160 kDa) is a glycoprotein and has several subunits. The beef-allergic patients showed strong reactivity to the approximately 200 kDa and approximately 60 kDa components of pure BGG by immunoblotting. Inhibition-ELISA showed that pure BGG preparations strongly inhibited the binding of sera from the beef-allergic patients to the beef extract. These results suggest that the approximately 200 kDa, approximately 67 kDa and approximately 60 kDa components in the beef extract had strong allergenicity: approximately 67 kDa was BSA, and approximately 200 kDa and approximately 60 kDa were presumably aggregated BGG and it's heavy chain, respectively.     

Immunodominance of conformation-dependent B-cell epitopes of protein antigens

Immunodominance of conformational epitopes over linear ones in four proteins was quantified making use of the B-cell hybridoma technology. The proteins were immunized in their native forms into BALB/c mice, and clonal frequencies of B-cell hybridomas that produce antibodies to the native and denatured forms were determined, using ELISA and immunoblotting. All 16 monoclonal antibodies (mAbs) to Porphyromonas gingivalis fimbria were suggested to recognize conformational epitopes expressed by the oligomer. Ten out of 14 mAbs to Serratia marcescens fimbria and 13 of 15 mAbs to hen lysozyme were also specific to their conformational epitopes. In contrast, all 18 mAbs to a surface protein of Streptococcus mutans, termed PAc, reacted to both the native and denatured forms, thereby indicating the immunodominance of linear epitopes in this protein. The results suggest that B-cell epitopes of proteins possessing stable tertiary or quaternary structures are predominantly expressed by the higher-order structures.     

Toxoplasma gondii: ultrastructural localization of specific antigens and inhibition of intracellular multiplication by monoclonal antibodies

This experiment was focused on the characterization of anti-Toxoplasma monoclonal antibodies (mAbs) and the effect of mAbs on the parasite invasion of mouse peritoneal macrophages. Twenty eight mAbs including M110, M556, R7A6 and M621 were characterized by Ab titer, immunoglobulin isotyping and western blot pattern. Antibody titer (optical density) of 4 mAbs, M110, M556, R7A6 and M621, were 0.53, 0.67, 0.45 and 0.39 (normal mouse serum; 0.19) with the same IgG1 isotypes shown by Enzyme-linked immunosorbent assay (ELISA). Western blot analysis showed that M110, M556, R7A6 and M621 reacted with the 33 kDa (p30), 31 kDa (p28), 43 kDa and 36 kDa protein. Immunogold labelling of mAbs M110, M556, R7A6 and M621 reacted with the surface membrane, dense granules and parasitophorous vacuolar membrane (PVM), rhoptries and cytoplasm of tachyzoite, respectively. For in vitro assay, preincubation of tachyzoites with four mAbs, M110, M556, R7A6 and M621 resulted in the decrease of the number of infected macrophages (p < 0.05) and the suppression of parasite multiplication at 18 h post-infection. Four monoclonal antibodies including M110 (SAG1) were found to have an important role in the inhibition of macrophage invasion and T. gondii multiplication in vitro, and these mAbs may be suitable for vaccine candidates, diagnostic kit and for chemotherapy.     

Characterization of monoclonal antibodies against human parvovirus B19

Eleven hybridoma cell lines producing mouse monoclonal antibodies (mAbs) against human parvovirus B19 were established. Their specificity was as follows. Approximately 5% of fetal erythroid cells inoculated with B19 reacted with all the mAbs and with anti-B19 positive human serum, but not with negative serum by indirect double immunofluorescence staining. All the mAbs recognized both VP-1 (84 kDa) and VP-2 (58 kDa) capsid proteins of B19 virions propagated in vitro and in vivo by Western blotting, and immunoprecipitated B19 virions.     

Heat-shock cognate protein (hsc71) and related proteins in mouse spermatogenic cells

A monoclonal antibody (13D3) has been developed that recognizes a 71 kilodalton (71 kDa) protein on two-dimensional immunoblots of proteins extracted from a mixture of mouse spermatogenic cells (mainly pachytene spermatocytes and spermatids). This protein was shown by immunoblotting and adenosine triphosphate (ATP)-binding characteristics to be identical to a 71 kDa mouse heat-shock cognate (hsc) protein, hsc71, present in 3T3 cells. Along with a 70 kDa heat-shock inducible protein (hsp70), and a 74 kDa heat-shock cognate protein (hsc74), hsc71 is a product of the mouse HSP70 multigene family. Although antibody 13D3 reacted strongly with hsc71, it reacted only faintly with hsp70 in 3T3 cells, and not at all with hsc74 or a germ cell-specific hsp70-like protein (P70) on immunoblots of mixed germ cells. Antibody 13D3 is unique among known antibodies in its pattern of reaction with these heat-shock proteins. In immunofluorescence studies on isolated germ cells, 13D3 reacted uniformly with the cytoplasm of pachytene spermatocytes, round spermatids, and residual bodies, but only with the midpiece of spermatozoa. Antibody 13D3 recognizes other proteins in addition to hsc71 on two-dimensional immunoblots of condensing spermatids and spermatozoa. Two of the proteins (70 kDa/pI 6.4 and 70 kDa/pI 6.5) were present in condensing spermatids and spermatozoa, and another protein (69 kDa/pI 7.0) was detected only in spermatozoa. The new proteins also were recognized by monoclonal antibody 7.10, which reacts specifically with hsp70, hsc71, hsc74, and P70. Although [35S]methionine was incorporated into the new proteins in condensing spermatids, hsc71, hsc74, and P70 were not labeled. These results suggest that unique heat-shock proteins are synthesized late in spermatogenesis.     

Plasmodium vivax:A Monoclonal Antibody Recognizes a Circumsporozoite Protein Precursor on the Sporozoite Surface

Gonzalez-Ceron, L., Rodriguez, M. H., Wirtz, R. A., Sina, B. J., Palomeque, O. L., Nettel, J. A., and Tsutsumi, V. 1998.Plasmodium vivax:A monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.Experimental Parasitology90, 203–211. The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells.Plasmodium vivaxCS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with otherPlasmodiumspecies, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with allP. vivaxsporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.     

Clathrin-Coated Vesicle Subtypes in Mammalian Brain Tissue: Detection of Polypeptide Heterogeneity by Immunoprecipitation with Monoclonal Antibodies

A panel of monoclonal antibodies (mAbs) was developed to identify polypeptides sorted in subtypes of brain coated vesicles (CVs) and to separate these by immunoprecipitation. The corresponding antigen of some of the mAbs elicited by CV components was present also in synaptosomal plasma membrane, synaptic vesicles, or microsomes. On immunoblots the mAbs reacted with constitutive brain CV proteins, with cargo molecules, and with a novel CV component that interacts with the actin cytoskeleton. Analysis of radioiodinated brain CVs immunoprecipitated with a tubulin antibody revealed that all brain CVs contained tubulin. The mAb A-7C11 recognized a 40-kilodalton (kDa) polypeptide on the clathrin coat and immunoprecipitated one-quarter of the total brain CVs. The mAb S-11D9 reacted with a 44-kDa antigen and immunoprecipitated 25% of the CVs. This antigen (44 kDa) was present in synaptic vesicles and synaptosomal membrane as well. Moreover, this mAb (S-11D9) reacted with a polypeptide of 56 kDa detected only in synaptosomal membrane. A mAb (C-10B2) that reacted with one of the clathrin light chains (LCb) immunoprecipitated 90% of the brain CVs. One of the mAbs immunoprecipitated a CV subtype that displayed a reversed ratio of the clathrin LCs (LCa greater than LCb). Each of the mAbs yielded different immunofluorescent staining patterns of vesicles in culture cell types that included nerve growth factor-differentiated PC12 cells, neuroblastoma cells, and Madin Darby bovine kidney cells. The data suggest that in brain tissue there is a heterogeneous population of CVs with different polypeptide compositions and subcellular distributions and that each of these subtypes performs a different role in nerve cells.     

Detection of phenylalanine hydroxylase protein in human platelets. Immunochemical detection

A protein reactive with anti-phenylalanine hydroxylase monoclonal antibody PH8 has been recovered from human platelet extracts. Two bands corresponding to molecular masses of about 60 kDa and 55 kDa were revealed by immunoblotting after electrophoresis according to Laemmli. Using the same antibody, a single band with a molecular mass of 60 kDa was demonstrated in extracts from human pineal gland; two similar antigens were found in human liver extracts and no antigen was found in adrenal gland extracts. Monoclonal antibodies, PH1 and PH3, did not react with these antigens during immunoblotting. Monoclonal antibodies, PH7 and PH9, reacted with the 55 kDa antigen in platelet extracts. The antigen content in platelet extracts was measured by ELISA with monoclonal antibodies relative to its content in the liver. The antigen content in platelet extracts was about 100 times as low as that in liver extracts and amounted to 100 ng/mg of protein. These findings suggest that the phenylalanine hydroxylase antigen is present in human platelets.     

Characterization of monoclonal antibodies against Gnathostoma nipponicum

Monoclonal antibodies (mAbs) were produced against the proteins of advanced third-stage larvae (AdL3) of Gnathostoma nipponicum. Six mAbs (Gn2C3, Gn2H3, Gn4C3, Gn4E9, GnSH1, and Gn10B7) were obtained as determined by enzyme-linked immunosorbent assay (ELISA). Gn4E9 and GnSH1 seemed to be genus-specific, as they did not cross-react with Anisakis sp., Dirofilaria immitis, Gongylonema pulchrum, Toxocara canis, Trichinella sp., Trichuris vulpis, Metagonimus sp., or Spirometra erinaceieuropaei by ELISA. Immunohistochemistry showed that Gn2C3, Gn4E9, and Gn5H1 reacted strongly with the central esophagus; Gn2H3 reacted with cuticle,muscle, intestine, and the cervical sac; and Gn4C3 and Gn10B7 reacted with cuticle, muscle, esophagus, intestine, and the cervical sac of AdL3. In Western blotting analysis, Gn2C3, Gn4E9, and Gn5H1 reacted to 60-, 53-, 46-, and 41-kDa proteins; Gn4C3 reacted to the AdL3 protein of G. nipponicum (>42 kDa). Moreover, proteins purified using a mAb Gn4E9 immunoprecipitation method (sizes 60-, 53-, 46-, and 41-kDa) were used as antigens in ELISAs. A significant difference (P < 0.01) was shown between mouse sera infected with G. nipponicum and sera infected with Trichnella sp. or not infected. These results provide a rationale for evaluating esophageal proteins for the development of diagnostic methods for detecting G. nipponicum or Gnathostoma sp. infections.     

Anti-idiotypic antibodies to a polyomavirus monoclonal antibody recognize cell surface components of mouse kidney cells and prevent polyomavirus infection.

Anti-idiotypic antibodies have been successfully used to identify and isolate the receptor for several cell ligands. To prepare an immunologic probe for identification of the polyomavirus receptor on mouse kidney cells, polyclonal antisera against antipolyomavirus antibodies were prepared in rabbits. Fab fragments of the previously characterized monoclonal antibody E7, which neutralizes polyomavirus infection, were used for immunization (S. J. Marriott and R. A. Consigli, J. Virol. 56:365-372, 1985). Sera containing the greatest anti-idiotype activity were identified by enzyme-linked immunosorbent assay (ELISA) and purified by a series of affinity columns. The anti-idiotypic antibodies recognized the E7 idiotope in an ELISA, and anti-idiotype binding could be inhibited by preincubation of E7 monoclonal antibody with polyomavirus virions. When mixed with anti-idiotype immunoglobulin G (IgG), E7 was no longer capable of binding or immunoprecipitating polyomavirus virions or neutralizing polyomavirus infection. Direct immunofluorescence showed anti-idiotype IgG reactivity with a cell surface component of mouse kidney cells. Anti-idiotype F(ab')2 effectively competed with polyomavirus for binding to mouse kidney cells and displayed binding kinetics similar to those of polyomavirus. Virus infection of mouse kidney cells was blocked in a dose-dependent manner following treatment of the cells with anti-idiotype IgG. The anti-idiotype identified several proteins (95, 50, and 24 to 31 kilodaltons) in an immunoblot of mouse kidney cell membrane proteins but reacted predominantly with a single 50-kilodalton protein in a radioimmunoassay. The anti-idiotype failed to react with virus proteins in three assays, including ELISA, immunoprecipitation, and immunoblotting. The implications of this work for future identification and characterization of the polyomavirus receptor on mouse kidney cells are discussed.     

Structural disulfide bonds in the Bacillus thuringiensis subsp. israelensis protein crystal.

We examined disulfide bonds in mosquito larvicidal crystals produced by Bacillus thuringiensis subsp. israelensis. Intact crystals contained 2.01 X 10(-8) mol of free sulfhydryls and 3.24 X 10(-8) mol of disulfides per mg of protein. Reduced samples of alkali-solubilized crystals resolved into several proteins, the most prominent having apparent molecular sizes of 28, 70, 135, and 140 kilodaltons (kDa). Nonreduced samples contained two new proteins of 52 and 26 kDa. When reduced, both the 52- and 26-kDa proteins were converted to 28-kDa proteins. Furthermore, both bands reacted with antiserum prepared against reduced 28-kDa protein. Approximately 50% of the crystal proteins could be solubilized without disulfide cleavage. These proteins were 70 kDa or smaller. Solubilization of the 135- and 140-kDa proteins required disulfide cleavage. Incubation of crystals at pH 12.0 for 2 h cleaved 40% of the disulfide bonds and solubilized 83% of the crystal protein. Alkali-stable disulfides were present in both the soluble and insoluble portions. The insoluble pellet contained 12 to 14 disulfides per 100 kDa of protein and was devoid of sulfhydryl groups. Alkali-solubilized proteins contained both intrachain and interchain disulfide bonds. Despite their structural significance, it is unlikely that disulfide bonds are involved in the formation or release of the larvicidal toxin.     

A panel of monoclonal antibodies recognizing GPI-anchored ADP-ribosyltransferase ART4, the carrier of the Dombrock blood group antigens

ART4 (CD297) is a member of the family of toxin-related ADP-ribosyltransferases (ARTs) and is the carrier of the Dombrock blood group alloantigens (Do). Two mouse monoclonal antibodies (MIMA-52 and MIMA-53), and two rat monoclonal antibodies (N0NI-B4 and NONI-B63) were obtained following immunization of mice with human Do/ART4-transfected cells and of rats with human Do/ART4 cDNA, respectively. All four mAbs recognize Do/ART4-transfected Jurkat cells but not untransfected cells by FACS analysis. Staining of Do/ART4-transfected cells by these mAbs was reduced following treatment of cells with PI-PLC, confirming that Do/ART4 is anchored in the cell membrane by linkage to glycosylphosphatidylinositol as predicted from its amino acid sequence. The four mAbs did not react with Gy(a-) (Dombrock null) erythrocytes but agglutinated other red blood cells. By flow cytometric analysis, all mAbs reacted prominently with erythrocytes, and weakly with peripheral blood monocytes and splenic macrophages, but not with B-lymphocytes or T-lymphocytes. The mAbs reacted weakly also with human umbilical vein endothelial cells and the basophilic leukemia KU-812. Immunohistology revealed staining of epithelia and endothelia on sections of tonsils. In FACS analyses NONI-B4 competed with MIMA-52 for binding to Do/ART4-transfected cells and erythrocytes, whereas NONI-B63 competed with MIMA-53. Neither of the mAbs reacted with mouse ART4-transfected cells, but NONI-B63 and MIMA-53 did react with a mouse/human ART4 chimera, indicating that the epitope recognized by these mAbs lies in the C-terminal half of the protein.