Identification of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1.

Hybrid arrest of in vitro translation was used to localize the region of the herpes simplex virus type 1 genome encoding the 65-kilodalton DNA-binding protein (65KDBP) to between genome coordinates 0.592 and 0.649. Knowledge of the DNA sequence of this region allowed us to identify three open reading frames as likely candidates for the gene encoding 65KDBP. Two independent approaches were used to determine which of these three open reading frames encoded the protein. For the first approach a monoclonal antibody, MAb 6898, which reacted specifically with 65KDBP, was isolated. This antibody was used, with the techniques of hybrid arrest of in vitro translation and in vitro translation of selected mRNA, to identify the gene encoding 65KDBP. The second approach involved preparation of antisera directed against oligopeptides corresponding to regions of the predicted amino acid sequence of this gene. These antisera reacted specifically with 65KDBP, thus confirming the gene assignment.     

Biological properties of antibodies against rat adipocyte intrinsic membrane proteins. Dependence on multivalency for insulin-like activity.

Antisera from rabbits injected with rat adipocyte plasma membranes or intrinsic proteins from such membranes, obtained by a dimethylmaleic anhydride extraction step, mimicked the action of insulin on both glucose transport and lipolysis in intact adipocytes. Biological activity in both types of antisera was mediated by immunoglobulin binding to one or more intrinsic proteins of the adipocyte plasma membrane since fat cells were unresponsive to all antisera absorbed with dimethylmaleic anhydride-extracted membranes. Acid treatment of immunoprecipitates released antibodies which activated glucose uptake and reacted with solubilized adipocyte membranes on immunodiffusion plates. The biologically active immunoglobulin preparations failed to form immunoprecipitin lines when tested against membranes from brain, liver, lung, muscle, kidney, and spleen. Insulin-sensitive glucose uptake in rat soleus muscle did not respond to the antisera. The antibodies activated hexose uptake into fat cells and reacted with solubilized adipocyte membranes on immunodiffusion plates when rat or mouse adipocytes were studied, but not when monkey fat cells were used. The anti-membrane antibody preparations readily activated hexose uptake in trypsinized fat cells which had lost the capacity to bind or respond to insulin. These data are consistent with the concept previously proposed (Pillion, D.J., and Czech, M.P. (1978) J. Biol. Chem. 253, 3761-3764) that the anti-membrane immunoglobulins do not interact with the insulin binding site of the insulin receptor. Monovalent Fab fragments of the biologically active antisera, prepared by papain digestion of the native anti-membrane immunoglobulins, were ineffective in enhancing glucose uptake in adipocytes. However, biological activity of the anti-membrane Fab fragments was restored by the addition of goat anti-rabbit Fab antisera to cells treated with the Fab fraction. Anti-rabbit Fab antisera alone or in combination with Fab fragments prepared from control rabbit sera exhibited no biological activity. These results demonstrate that the ability of anti-membrane antisera to mimic the biological activity of insulin on isolated fat cells is critically dependent on immunoglobulin binding to one or more intrinsic plasma membrane proteins and the multivalent nature of immunoglobulin structure.     

大鼠精核蛋白抗血清的制备

用大鼠精核蛋白（Ｒａｔ　Ｐｒｏｔａｍｉｎｅ,ＲＰ）－核糖核酸（ＲＮＡ）复合物（ＲＰ－ＲＮＡ　Ｃｏｍｐｌｅｘｅｓ）免疫大鼠,得到了特异的抗ＲＰ抗血清,并用Ｉｍｍｕｎｏｄｏｔｔｉｎｇ和Ｉｍｍｕｎｏｂｌｏｔｔｉｎｇ方法验证了其特异性。该抗血清和ＲＰ有特异的反应,并和哺乳动物小鼠和羊的精核蛋白有一定程度交叉反应,而与体细胞类型核蛋白（Ｈ１,Ｈ２ａ,Ｈ２ｂ,Ｈ３,Ｈ４）无交叉反应。并对制备该抗血清的意义予以讨论。     

Specific and shared idiotypes found on hybridoma anti-DNA autoantibodies derived from rheumatoid arthritis and systemic lupus erythematosus patients

The idiotype determinants found on hybridoma anti-DNA autoantibodies produced from the fusion of peripheral blood lymphocytes from 13 systemic lupus erythematosus (SLE) and five rheumatoid arthritis (RA) patients with the GM 4672 human lymphoblastoid line were analyzed. A total of 47 SLE and 21 RA hybridomas were studied, of which 26 SLE and 10 RA produced anti-DNA autoantibodies. Rabbit antisera, raised to six of the SLE hybridoma anti-DNA IgM antibodies, were rendered idiotype specific by multiple absorptions on human IgM and IgG immunoabsorbent columns. In direct binding radioimmunoassays, all six anti-idiotype antisera reacted specifically with the anti-DNA antibody used as immunogen. In competition studies, five anti-idiotype antisera were able to inhibit the binding of their homologous idiotype to DNA-coated tubes. In addition, DNA and polynucleotides inhibited the binding of the five idiotypes to anti-idiotype-coated tubes, suggesting that these anti-idiotypes react with idiotype determinants located within the antigen-combining sites of the anti-DNA antibody molecules. Shared idiotypes were detected among the 68 hybridoma antibodies by direct binding studies on anti-idiotype-coated tubes. Our results revealed that 58% (21/36) of the anti-DNA antibodies and 16% (5/32) of the non-DNA-binding antibodies reacted with at least one anti-idiotype serum. Five anti-idiotype antisera reacted only with hybridoma anti-DNA antibodies from SLE patients. The other anti-idiotype antiserum reacted with both SLE- and RA-derived hybridoma anti-DNA and non-DNA-binding antibodies. These studies indicate that some anti-idiotype antisera may detect specific idiotypes found only on SLE-derived anti-DNA auto-antibodies, whereas other antisera detect shared idiotypes found on both RA and SLE DNA-binding and non-DNA-binding antibodies.     

Synthesis In Vitro of Type 5 Adenovirus Capsid Proteins

Reaction mixtures containing cytoplasmic extracts and ribosomal fractions prepared from KB cells infected with type 5 adenovirus were able to carry out incorporation of amino acids into protein. The in vitro product included proteins which reacted specifically with antisera to adenovirus capsid proteins; in control experiments with extracts from uninfected cells, no reactions with the antisera were found. The viral proteins were synthesized in vitro on small polyribosomes, were released from them, and significant numbers of the free polypeptides were assembled in vitro into multimeric adenovirus capsid structures.     

Development, Characterization, and Use of Monoclonal and Polyclonal Antibodies against the Myxosporean, Ceratomyxa shasta

Both monoclonal and polyclonal antisera were produced against Ceratomyxa shasta. Ascites containing trophozoites of the parasite was collected from infected fish and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted specifically with trophozoite and sporoblast stages but did not react with C. shasta spores by either indirect fluorescent antibody techniques or in Western blots. This indicates that some C. shasta antigens are specific to certain life stages of the parasite. Polyclonal antiserum was produced in a rabbit by injecting a spore protein electro-eluted from an SDS-polyacrylamide gel. This antiserum reacted with both trophozoites and spores by indirect fluorescent antibody techniques and in Western blots. All antisera were tested for cross-reactivity to trout white blood cells, a contaminant of the ascites, and to other myxosporea. Two monoclonal antibodies reacted with white blood cells and myxosporea of the genera Sphaerospora and Myxobilatus. One hybridoma produced antibodies of high specificity for C. shasta pre-spore stages. This is the first report of a monoclonal antibody produced against a myxosporean parasite.     

In vitro characterization of anti-glucosylceramide rabbit antisera

Glucosylceramides (GlcCer) are biosynthetic precursors of glycosphingolipids. They are widely distributed in biological systems where they exhibit numerous biological functions. Studies on the localization of glucosylceramides in different tissues have used biochemical methods only since specific antibodies against GlcCer were not previously available. We have characterized two commercially available rabbit antisera which were prepared against GlcCer of plant origin (1-O-(beta-D-glucopyranosyl)-N-acyl-4-hydroxysphinganine; GlcCer-3) or human origin (1-O-(beta-D-glucopyranosyl)-N-acyl-sphingosine; GlcCer-2) and claimed to be specific for GlcCer. The antisera were also able to detect specifically GlcCer species in crude lipid extracts from human epidermis after separation by thin-layer chromatography. The reagents are sensitive since both antisera reacted at dilutions higher than 1:500 with their homologous antigen in the nanogram range in thin layer immunostaining or dot-blot assays. The antisera are specific for GlcCer although they did not differentiate between GlcCer-2 and GlcCer-3 containing sphingosine or 4-hydroxysphinganine. The antisera also reacted with N-stearoyl-DL-dihydroglucocere-broside indicating that the naturally occurring structural variations in the amino alcohol moiety are not determining the specificity. No crossreactivity was observed with other mono- or diglycosylceramides (galactosylceramides, lactosyl-ceramide), free ceramides or structurally unrelated lipids (cholesterol, sphingomyelin, or phospholipids). Therefore, the glycosylmoiety seems to represent the major antigenic determinant. Finally, the antisera also proved to be useful for the immunohistochemical localization of GlcCer in human epidermis by which earlier biochemical data on the distribution of GlcCer in the various epidermal layers were confirmed.     

Serological and biological characterisation of isolates of barley yellow dwarf virus in Sweden in 1983

In 1983, cereal plants showing symptoms of barley yellow dwarf virus (BYDV), collected from 15 localities in Sweden, were tested for BYDV using enzyme-linked immunosorbent assay (ELISA). Antisera against two Swedish isolates of BYDV were used, a mild isolate (27/77) transmitted specifically by Sitobion avenae and a severe one (39/78) transmitted mainly by Rhopalosiphum padi. No virus was detected in 57 of 607 plants of oats and barley tested. Of the 550 plants in which virus was detected, 366 were infected with viruses similar to isolate 27/77, 116 with viruses similar to 39/78 and the remaining 68 reacted strongly with both antisera. When tested, the latter isolates were shown to be mixtures. Thirty-nine selected samples were also tested with antisera against the USA isolates RPV, RMV, MAV and PAV, and for transmission by S. avenae and R. padi. Twenty-six of these samples were transmitted specifically by S. avenae, one was transmitted only by R. padi and the remaining 12 samples were shown to be infected with a mixture of an S. avenae-specific isolate and one transmitted mainly by R. padi. Antisera against PAV and MAV each detected all isolates tested and the results were very similar to those with the antisera to the 39/78 and 27/77 isolates, respectively. None of the field isolates reacted with antisera against RMV or RPV. It was concluded that 1983 was an epidemic year for BYDV in Sweden and that isolates specifically transmitted by S. avenae predominated. Symptoms of infection by these isolates on oat plants ranged from mild to severe.     

The serological specificity of S alleles of homozygous incompatible lines of the marrow-stem kale (Brassica oleracea var.acephala DC.)

The preparation of antisera against S alleles of homozygous incompatible lines of the marrow-stem kale (Brassica oleracea var.acephala DC.) is described. The antisera obtained reacted specifically with homologous antigens on using the serological method of double diffusion into agar. The results confirmed the specificity of S alleles of incompatible lines of the marrow-stem kale.     

Attempts to probe the antigens and protective immunogens of Trichostrongylus colubriformis in immunoblots with sera from infected and hyperimmune sheep and high- and low-responder guinea pigs

Comparison of antisera from sheep during primary infection and following vaccination and challenge with Trichostrongylus colubriformis, with antisera obtained following primary infection of high- and low-responder guinea pigs, failed to reveal different antigenic patterns in proteins separated from fourth stage larval extracts by two-dimensional electrophoresis and probed by the immunoblot technique.Generally, serum IgG reacted specifically with worm antigens of mol. wt greater than 94,000, whereas protection against challenge infection was elicited most effectively in the guinea pig by fractions in the 67,000–94,000 range.Most distinct separations of larval proteins by SDS-polyacrylamide gel electrophoresis were obtained by extraction of live larvae and the extracts used within 2–3 days.     

Detection of T-cell surface determinants in three Marek's disease lymphoblastoid cell lines.

The T-cell surface antigens on three lymphoblastoid cell lines, MOB1, MOB-2 and MSB-1, derived from Marek's disease lymphomas were examined by the cytotoxicity test and the indirect membrane immunofluorescent test. These cell lines reacted specifically with antisera prepared against chicken thymus cells, although their reactivities were less than that of typical thymus cells. These three cell lines were of thymus origin.     

Identification of human erythrocyte blood group antigens on the C3b/C4b receptor.

The Knops/McCoy (Kn/McC) human erythrocyte blood group system belongs to the category of blood group Ag that generate so-called "high titer low avidity" antibodies in immunized transfusion recipients. Screening of red cells lacking certain high titer low avidity Ag demonstrated markedly diminished CR1 expression on McC(d-) and Kn/McC "null" (Kn(a-)McC(a-b-c-d-e-f-] erythrocytes. Additional testing by other methods confirmed these data, and biochemical assays demonstrated no detectable immunoreactive CR1 protein in membranes from Kn/McC null red cells. Human antisera to various Kn/McC Ag were then used to demonstrate that many of these antisera could be used to isolate a protein of identical m.w. to that isolated from the same cells using murine mAb CR1 antisera. Finally, protein isolated by using murine mAb anti-CR1 reacted specifically with anti-Kn/McC antibodies, demonstrating the identity of the Kn/McC and CR1 proteins. Thus, CR1 protein bears the human erythrocyte Kn/McC blood group Ag.     

Production and characteristics of antisera to Spiroplasma citri and clover phyllody-associated antigens derived from plants

Antisera were produced to clover phyllody- and Spiroplasma citri-associated antigens partially purified from infected Vinca rosea plants. Separate antisera were made to ‘membrane fraction’ (MF) preparations comprising the resuspended pellet obtained by high speed centrifugation, and to ‘soluble fraction’ (SA) preparations, comprising the supernatant from high speed centrifugation concentrated by freeze-drying. All antisera showed considerable activity against normal plant antigens but after cross-absorption with extracts of healthy plants the MF antisera were used in F(ab')₂based ELISA tests to detect S. citri- or clover phyllody-associated antigens in infected plants. The ‘clover phyllody’ antiserum also reacted specifically with extracts of clover plants with phyllody, and with naturally-infected strawberry plants showing symptoms of green petal disease. Both the ‘clover phyllody’ and S. citri antisera were specific for their respective homologous antigens. No cross-reactions were observed in heterologous tests or between either antiserum and extracts of V. rosea infected with various MLOs obtained from different host plants.     

Identification and characterization of maize pathogenesis-related proteins. Four maize PR proteins are chitinases

Eight pathogenesis-related proteins extractable at pH 2.8 were found to accumulate in maize leaves after mercuric chloride treatment or brome mosaic virus infection. These proteins were called PRm (pathogenesis-related maize) proteins. Seven PRm proteins were purified to homogeneity by preparative polyacrylamide gel electrophoresis and their amino acid compositions determined. Estimated molecular weights in SDS-containing gels were: PRm 1 14.2 kDa; Prm 2 16.5 kDa; PRm 3 and PRm 4 25 kDa; PRm 6b 30.5 kDa; PRm 6a 32 kDa; PRm 7 34.5 kDa. Antisera raised against either PRm 3 or PRm 4 reacted specifically each with PRm 3 or PRm 4. Antisera raised against PRm 6b reacted with PRm 6b as well as with PRm 6a and antisera against PRm 7 reacted with PRm 7 and PRm 5. Tobacco anti-PR 1b antisera reacted with maize PRm 2.Chitinase (poly[1,4-(N-acetyl--D-glucosamide)]glycanhydrolase, EC 3.2.1.14) activity was found for PRm 3, PRm 4, PRm 5, and PRm 7.     

Class-specific epitopes detected by polyclonal antibodies against the secretory products of the subcommissural organ of the dogfish Scyliorhinus canicula

We have raised antisera against extracts of the subcommissural organ (SCO) of the dogfish, Scyliorhinus canicula L. Brains of 2900 specimens were collected in acetone, and the region containing the SCO and posterior commissure was removed and extracted in three different media. Antisera against these crude extracts were raised in rats and rabbits. Sequential absorptions of the antisera with extracts from different regions of the dogfish brain were performed to eliminate unwanted antibodies. When used to immunostain sections of the whole central nervous system of the dogfish, these purified antisera reacted selectively with the SCO-Reissner's fiber complex. An antiserum against bovine Reissner's fiber was also used. The antisera against the dogfish SCO and bovine Reissner's fiber showed the same staining pattern in the SCO and the Reissner's fiber of the dogfish. For comparative purposes, the brains of 15 vertebrate species from all vertebrate classes were immunostained with both antisera. The anti-dogfish SCO serum reacted with the SCO of the dogfish and that of other phylogenetically related elasmobranch species. Neither the SCO of a primitive elasmobranch species, Heptranchias perlo, nor the SCO of the other classes of vertebrates reacted with the anti-dogfish SCO serum. However, the antiserum against bovine Reissner's fiber reacted with the SCO of all the investigated species. It is concluded that some epitopes (or compounds) in the secretory material of the SCO are class-specific, whereas others are conserved and are synthesized by the SCO in most vertebrate species.     

Identification of Immunoreactive Material in Mammoth Fossils

The fossil record represents a history of life on this planet. Attempts to obtain molecular information from this record by analysis of nucleic acids found within fossils of extreme age have been unsuccessful or called into question. However, previous studies have demonstrated the long-term persistence of peptides within fossils and have used antibodies to extant proteins to demonstrate antigenic material. In this study we address two questions: Do immunogenic/antigenic materials persist in fossils? and; Can fossil material be used to raise antibodies that will cross-react with extant proteins? We have used material extracted from a well-preserved 100000-300000-year-old mammoth skull to produce antisera. The specificity of the antisera was tested by ELISA, western blotting, and immunohistochemistry. It was demonstrated that antisera reacted specifically with the fossils and not the surrounding sediments. Reactivity of antisera with modern proteins and tissues was also demonstrated, as was the ability to detect evolutionary relationships via antibody-antigen interactions. Mass spectrometry demonstrated the presence of amino acids and specific peptides within the fossil. Peptides were purified by anion-exchange chromatography and sequenced by tandem mass spectrometry. The collagen-derived peptides may have been the source of at least some of the immunologic reactivity, but the antisera identified molecules that were not observed by mass spectrometry, indicating that immunologic methods may have greater sensitivity. Although the presence of peptides and amino acids was demonstrated, the exact nature of the antigenic material was not fully clarified. This report demonstrates that antibodies may be used to obtain information from the fossil record.     

Detection of cross-reactive determinants shared by human monoclonal IgM reacting with myelin-associated glycoprotein

Monoclonal IgM from patients with peripheral neuropathy frequently have anti-myelin-associated glycoprotein (MAG) activity. We investigated the idiotypes of 10 monoclonal anti-MAG by using rabbit polyclonal antisera. Three groups of anti-idiotypic antisera could be distinguished. Four sera reacted only with the immunizing protein. Two sera reacted with a single other anti-MAG IgM in addition to the immunizing one. Immunoenzymatic studies showed that these two couples of anti-MAG IgM reacted identically with 100% cross-inhibition, indicating that the whole set of idiotypes identified by the rabbit antiserum was present on both IgM antibodies. The four other anti-idiotypic sera reacted with the homologous IgM, as well as with most of the other anti-MAG IgM. In contrast to the previous antisera the binding of these four sera to the homologous IgM could not be inhibited by other cross-reacting anti-MAG IgM. However, when a heterologous IgM was used for coating, these antisera with one exception showed complete cross-inhibition. The absence of inhibition by purified MAG of the patient of the anti-idiotypic antisera sera suggests that these antibodies are most likely to be directed against framework determinants rather than against combining site epitopes.     

So-called antireceptor sera

Experiments in delayed type hypersensitivity transfer were carried out with the aim of studying the ability of rabbit antisera against peritoneal exudate cells of rats sensitized with bovine gamma globulin or rabbit kidney tissue antigen to block peritoneal exudate cells of guinea pigs. In the serological test the antisera prepared against the cells of sensitized rats and tentatively named "receptor antisera", reacted not only with the cells of these rats, respectively, but also with guinea pig cells. In hypersensitivity transfer experiments in guinea pigs receptor antisera showed a blocking effect on the transferred cells, making them incapable of transferring hypersensitivity, i. e. rabbit antisera against rat peritoneal exudate cells reacted with guinea pig cells. This interaction was specific: the blocking effect was manifested only when guinea pigs whose cells were used in the transfer were sensitized with the same antigen as the rats against whose cells the receptor antisera had been prepared. The control antisera, taken for the treatment of the transferred cells in the same doses as the receptor antisera, had no blocking effect on the cells.     

Expression of meningococcal epitopes in LamB of Escherichia coli and the stimulation of serosubtype-specific antibody responses

The class 1 outer membrane protein (OMP), a major variable surface antigen of Neisseria meningitidis, is a component of novel meningococcal vaccines currently in field trials. Serological variants of the protein are also used to serosubtype meningococci. Most of the amino acid changes that give rise to antigenic variants of the protein occur in two variable regions (VR1 and VR2) that are thought to form loops on the cell surface. The polymerase chain reaction (PCR) was used to amplify the nucleotide sequences encoding VR1 and VR2 from the chromosomal DNA of N. meningitidis strain M1080. These were cloned in frame into the lamB gene of the Escherichia coli expression vector pAJC264. Whole-cell enzyme-linked immunosorbent assays (ELISAs), using monoclonal antibodies, and SDS PAGE confirmed that, upon induction, strains of E. coli carrying these constructs expressed hybrid LamB proteins containing the N. meningitidis surface loops. These strains were used to immunize rabbits and the resultant polyclonal antisera reacted specifically with the class 1 OMP of reference strain M1080 (P1.7). Immunogold labelling of meningococcal cells and whole-cell dot-blot analyses with these antisera showed that the variable epitopes were exposed on the cell surface and confirmed that this approach could be used to obtain serosubtype-specific antisera. The binding profiles of the antisera were determined from their reactions with overlapping synthetic peptides and their reactivity compared with that of relevant serosubtype-specific monoclonal antibodies. This approach was used successfully to raise antisera against two other class 1 OMP VR2s. A fourth antiserum raised against a VR2, including the P1.1 epitope, was not subtype specific.     

Molecular cloning of an acrosomal sperm antigen gene and the production of its recombinant protein for immunocontraceptive vaccine

A monoclonal antibody, HS-63, which reacts specifically with a highly conserved sperm acrosome antigen, was shown to inhibit in vitro fertilization of mouse and human. The corresponding sperm antigen designated as MSA-63 was purified to homogeneity from mouse testes and used as an immunogen to generate polyclonal antisera in rabbits. The cDNA fragments of MSA-63 gene were cloned from mouse testis cDNA library by an immunoscreening method using polyclonal antisera specific for MSA-63. Using the established cDNA clone as a probe, the gene encoding for MSA-63 protein was found to be conserved among different mammalian species. Only one specific mRNA 1.5 kb in size was identified from the adult mouse testis among different mouse tissues. The recombinant fusion protein containing MSA-63 protein fragment was produced in Escherichia coli and used to immunize female mice. Similar to the original HS-63 monoclonal antibody, the antisera thus produced reacted only with the sperm acrosome and revealed significant inhibition to the in vitro fertilization of mouse oocytes. The results of this preliminary study suggest that it is feasible to mass produce sperm-specific antigens or their antigenic fragments by recombinant DNA technology for the development of sperm antigen-based immunocontraceptive vaccines.