Proteins of hepatitis B surface antigen.

Purified 22-nm forms of hepatitis B surface antigen (Hbsag) representing the three major antigenic subtypes (adw, ayw, and adr) were analyzed for their constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No consistent difference in either the number or relative distributions of the polypeptides was observed for the various subtypes. Seven polypeptides were designated as P-1 through P-7 in order of their decreasing mobilities. By comparison with protein standards, their molecular weights were estimated as 23, 29.5, 36, 41.5, 53.5, 72, and 97 thousand. The P-1 and P-2 components represented the major polypeptides; P-2 and P-5 might by glycoproteins, based on their reaction with periodic acid-Shiff reagent. Each polypeptide contains cysteine residues. HBSAg was radiolabeled with 3H or 14C by reductive methylation or iodinated with 125I by the chloramine-T or lactoperoxidase procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled HBSAg yielded patterns identical to those obtained with protein stain. Comparison of HBSAg labeled by the chloramine-T and lactoperoxide procedures indicated that there was no distinction between internal or external components within the 22-nm structure.     

Biophysical characterization of the adr subtype of hepatitis B antigen and preparation of anti-r sera in rabbits.

The biophysical properties of the adr subtype of HBSAg were determined and found to be identical to those of the adw and ayw phenotypes. These properties were used to purify the 22 nm spherical form of HBSAg/adr from the serum of a chronic antigen carrier by using zonal centrifuge techniques. The purified antigen was injected into 15 rabbits which were bled weekly to follow the development of antibodies to HBSAg by hemagglutination, and to the subtype determinants of HBSAg by counterelectrophoresis and agar gel diffusion (AGD) assays. From these data and two pilot studies to produce anti-w using an adw and an ayw antigen the relative immunogenicities of the various HBSAg determinants could be ranked as a greater than r greater than d greater than y greater than w. A selected pool of the sera from the rabbits immunized with HBSAg/adr was prepared as an interim reference reagent; when diluted 1:4 in saline and tested by AGD, it contains anti-a and anti-r without demonstrable anti-d or anti-normal human serum activity.     

乙型肝炎病毒表面抗原多肽图谱与亚型关系

用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western吸印法分析乙型肝炎病毒表面抗原(HBsAg)多肽组成,与蛋白标准品比较获得分子量23000～50000 8种多肽,用单一抗各亚型因子血清分析,证实a抗原决定簇存在于多种多肽中,d抗原决定簇主要由分子量23000的多肽组成,而y抗原决定簇则由分子量31000及34000的两种多肽组成。     

The demonstration of subtype (D or Y)-specific determinants on the surface of the presumed hepatitis B virus.

Dane particles isolated from the sera of HBsAg/ad and HBsAg/ay carriers were reacted with monospecific antibodies to the d and y subtype-specific determinants of HBsAg/. Dane particles from HBsAg/ad expressed the d determinant on their surfaces and those from HBsAg/ay sera contained the y specificity. Both complete (DNA-P and HBcAg) and defective (HBcAg alone) Dane particles expressed the subtype-specific determinants.     

Antibodies Specific for α-Subunit Subtypes of GABAA Receptors Reveal Brain Regional Heterogeneity

Abstract: Antisera were produced in rabbits against synthetic peptides based on subtype-specific regions of the cDNA sequences of the α1, α2, α3, and α4 (also termed α5) subunits of mammalian GABA_A receptors. The antigen peptides were chosen from the putative cytoplasmic loop between the proposed third and fourth membrane spanning helices; they were not only subtype-specific sequences, but also their hydrophilicity and predicted secondary structures suggested high potential antigenicity. In all cases, antipeptide antisera recognized on western blots the corresponding α-subunit polypeptide of the GABA_A receptors purified from bovine brain by benzodiazepine-affinity chromatography, and were able to immunoprecipitate binding activity from detergent-solubilized purified receptors. The four antisera each recognized a unique polypeptide, and only one, in the purified receptor, with α1, α2, α3, and α4 identified at 51, 52, 56, and 57 kDa, respectively. This represents the first identification of the α4 gene product on a gel. Both the relative amount of staining in immunoblots and the fraction of receptor binding that could be immunoprecipitated by saturating concentrations of each of the four subtypespecific antibodies varied in a consistent manner between receptors purified from different brain regions. Thus, cerebral cortex receptor contained all four α polypeptides on western blots, and significant activity could be precipitated by all four. Hippocampal receptor lacked α3 immunoreactivity on blotting and by immunoprecipitation; α1 was less, whereas both α2 and α4 were more abundant in hippocampus than in cortex by both techniques. Cerebellum receptor contained only α1 of the four α subunits tested, and the anti-α1 antibodies immunoprecipitated >90% of the binding activity. The variable amounts of staining and immunoprecipitation from the three brain areas by the four antisera demonstrate the presence of heterooligomeric receptor complexes with different α-subunit constituents in cortex, hippocampus, and cerebellum. The sum of cortical receptor activity precipitated individually by the four anti-α antisera was > 150%, indicating that some heterooligomers are likely to contain more than one class of α subtype, although most receptor complexes probably contain only one α subtype. These α-subunit subtype-specific antibodies should be useful in analyzing structure, function, and localization of GABA_A/benzodiazepine receptors in mammalian brain.     

Integration of different keratins into the same filament system after microinjection of mRNA for epidermal keratins into kidney epithelial cells

We have isolated poly (A)+ RNA, highly enriched in keratin mRNA from bovine muzzle epidermis, and injected it into epithelial cells of a different type, i.e., cultured kidney epithelial cells of the same (MDBK) or taxonomically distant (PtK2) species. Both recipient cell lines contain keratin polypeptides that are different from those present in epidermal cells. Using keratin subtype-specific antibodies in immunofluorescence and immunoelectron microscopy, we show that foreign keratin mRNAs when injected into a different type of epithelial cell can recruit polyribosomes and are translated together with the keratin mRNAs of the host cell. Foreign epidermal keratins are excluded from vimentin filaments and other structures but readily coassemble with the endogenous keratins and appear to be integrated into the meshwork of the preexisting kidney-type keratin filaments. Our observations indicate that different sets of keratin polypeptides from the same or different species can coassemble in the living cell into a common filament system. Thus we have developed a procedure that allows experimental alteration of the intermediate filament cytoskeleton within living epithelial cells.     

Parasite specific antibodies in three strains of mice after infection with Trypanosoma cruzi

Three inbred strains of mice (BALB/cJ, C3H/HeJ and NZB/BInJ) were infected with trypomastigotes of Trypanosoma cruzi. Sera were taken at different times after infection and radioimmunoprecipitation assays were used to detect antibodies against individual T. cruzi epimastigote and trypomastigote antigens. The mouse strains differed in regard to the spectrum of antibodies and the time after infection when the various epimastigote specific antibody species appeared. NZB mice had antibodies against at least 25 polypeptides ranging in molecular weight from 20,000 to 90,000 D at 3 wk after infection, and these persisted until at least 10 wk post-infection. C3H and BALB/c had antibodies against fewer than 5 antigens at 3 wk after infection; whereas by week 10, antibodies against at least 25 polypeptides were detected. C3H mice that were most susceptible to infection (but not NZB or BALB/c mice) had antibodies against a 25,000 D molecular weight epimastigote antigen. The antibody response against trypomastigote polypeptides was more uniform. Sera from all mouse strains at 3 wk after infection precipitated the same polypeptides and the radioimmunoprecipitation patterns did not change as a function of time after infection.     

Characterization of monoclonal antibodies that recognize band 4.5 polypeptides associated with nucleoside transport in pig erythrocytes.

Three monoclonal antibodies have been raised against partially purified band 4.5 polypeptides [Steck (1974) J. Cell Biol. 62, 1-19] from pig erythrocyte membranes. The antibodies were capable of binding to both intact pig erythrocytes and protein-depleted membrane preparations and recognized detergent-solubilized polypeptides from adult and neonatal pig erythrocytes that were photolabelled with [G-3H]nitrobenzylthioinosine (NBMPR), a potent specific inhibitor of nucleoside transport. The antibodies did not recognize polypeptides from neonatal pig erythrocytes that were photolabelled with the glucose-transport inhibitor [3H]cytochalasin B. Reactivity with polypeptides of apparent Mr 64,000 [10% (w/v) acrylamide gels] was demonstrated by Western-blot analysis. The antibodies recognized pig band 4.5 polypeptides after prolonged treatment with endoglycosidase F, a finding consistent with reactivity against polypeptide, rather than carbohydrate, determinants. Trypsin digestion of NBMPR-labelled protein-depleted pig erythrocyte membranes generated two labelled polypeptide fragments (Mr 43,000 and 26,000). Two of the antibodies recognized both fragments on Western blots, whereas the third bound to the larger, but not to the smaller, fragment. The antibodies had no significant effect on reversible binding of NBMPR to protein-depleted pig erythrocyte membranes and did not bind to NBMPR-labelled polypeptides in human, rabbit or mouse erythrocytes.     

Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II

A collection of 17 monoclonal antibodies elicited against the light-harvesting chlorophyll a/b protein complex which serves photosystem II (LHC-II) of Pisum sativum shows six classes of binding specificity. Antibodies of two of the classes recognize a single polypeptide (the 28- or the 26- kD polypeptides), thereby suggesting that the two proteins are not derived from a common precursor. Other classes of antibodies cross-react with several polypeptides of LHC-II or with polypeptides of both LHC-II and the light-harvesting chlorophyll a/b polypeptides of photosystem I (LHC-I), indicating that there are structural similarities among the polypeptides of LHC-II and LHC-I. The evidence for protein processing by which the 26-, 25.5-, and 24.5-kD polypeptides are derived from a common precursor polypeptide is discussed. Binding studies using antibodies specific for individual LHC-II polypeptides were used to quantify the number of antigenic polypeptides in the thylakoid membrane. 27 copies of the 26-kD polypeptide and two copies of the 28-kD polypeptide were found per 400 chlorophylls. In the chlorina f2 mutant of barley, and in intermittent light-treated barley seedlings, the amount of the 26-kD polypeptide in the thylakoid membranes was greatly reduced, while the amount of 28-kD polypeptide was apparently not affected. We propose that stable insertion and assembly of the 28-kD polypeptide, unlike the 26-kD polypeptide, is not regulated by the presence of chlorophyll b.     

Characterization of tonoplast polypeptides isolated from corn seedling roots

Tonoplast vesicles were isolated by discontinuous sucrose gradient centrifugation in the presence of Mg²⁺ from 5 day old corn (Zea mays L., Golden Cross Bantam) seedling roots. Marker enzyme assays indicated only a low degree of cross-contamination of tonoplast vesicles at the 10/23% (weight/weight) interface by other membrane components. Severalfold enrichment of tonoplast ATPase and pyrophosphatase was indicated in tonoplast fractions by dot blot studies with antibodies against an oat tonoplast ATPase and a mung bean tonoplast pyrophosphatase. Comparison of two-dimensional electrophoretic gels of tonoplast and microsomal membrane polypeptides revealed approximately 68 polypeptides to be specific to tonoplast by silver staining. Immunoblot analysis with antibodies against a tonoplast holoenzyme ATPase from oat roots revealed the presence of the 72, 60, and 41 kilodalton polypeptides in isolated tonoplast vesicles from corn roots. Affinity blotting with concanavalin A and secondary antibodies indicated the degree of glycosylation of tonoplast polypeptides, where 21 of 68 tonoplast-specific polypeptides contained detectable carbohydrate moieties. Salt and NaOH washes removed 38 of the tonoplast-specific polypeptides, indicating a peripheral association with the membrane. Thirteen of the peripheral polypeptides and eight of the integral polypeptides were identified as glycoproteins. This information on the polypeptide composition of the tonoplast of root cells will aid in gaining insight into the role of this membrane in controlling vacuolar functions.     

Occurrence of hepatitis and hepatitis B surface antigen in adult patients with acute leukemia.

Fifty-eight adult patients with acute leukemia were screened at the onset of the disease for hepatitis B antigen (HBSAg) in the serum, and during the course of the disease for the development of hepatitis B. One patient had a positive test for HBSAg by the radioimmunoassay technique only at the time leukemia was diagnosed; this patient had received transfusions some years before. In six patients icteric hepatitis B developed; five recovered completely and one died of leukemia during the course of hepatitis. All patients in whom hepatitis developed had received transfusions as a part of supportive therapy for leukemia. The hepatitis risk for patients who received transfusions of blood found to be negative for HBSAg by counterimmunoelectrophoresis was 0.26 percent per unit of blood administered.     

Identification of bovine sperm specific polypeptides reactive with antisperm antibodies.

The present study was taken to characterize molecular weights of sperm specific polypeptides antigenic to rabbits and calf with the aim to assess their immunoreactivity with IgG antibodies in sera from immuno-infertile cows. Seropositivity for antisperm IgG antibodies in 75 repeat breeder and 15 pregnant control cattle was tested by cellular ELISA using washed spermatozoa antigen from 4 bulls. Molecular weights of bovine sperm polypeptides antigenic to rabbit and calf were determined by 10% SDS-PAGE and Western blotting. Molecular weights of sperm peptides reactive with sera from immuno-infertile cows were also determined. Seropositivity of antisperm IgG antibodies for bull I, II, III and IV was 23.6, 14.6, 26.6 and 20%, respectively. A total of 16 polypeptides were discernible on gel. Out of these, 7 polypeptides were immunoreactive with sera from hyperimmunized rabbits as compared to 3 poly-peptides which reacted with sera from hyper-immunized calf. Only two polypeptides were reactive with sera from immuno-infertile cows. Variable number of sperm polypeptides and their immunoreactivity have been reported in different species. Antigenicity of different polypeptides in sperm needs further investigations.     

Monoclonal antibodies as probes for processing of yolk protein in the mosquito; production and characterization

We have produced a library of monoclonal antibodies against yolk proteins of the mosquito Aedes aegypti. After the initial screening, 45 hybridoma cell lines were selected and cloned. Immunoblot analysis revealed three groups of monoclonal antibodies. One group recognized a 200-kDa polypeptide, the second a 68-kDa, and the third both of these polypeptides. While the affinity of binding by different antibodies varied widely, all monoclonal antibodies recognized these polypeptides only in extracts from vitellogenic fat bodies and ovaries. The antibodies were further characterized by video-enhanced immunofluorescence, which also showed that both yolk polypeptides originated in the fat body and accumulated in the oöcytes. The immunolocalization in trophocytes of the fat body suggested that monoclonal antibodies may recognize different stages of the secretory pathway of yolk polypeptides. Similar analysis of oöcytes indicated that our panel of antibodies recognizes different steps of processing of both 200-kDa and 68-kDa polypeptides, beginning with internalization by the oöcyte and ending with the final crystalline form in mature yolk bodies.     

Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products

Hybridomas secreting monoclonal antibodies specific for the adenovirus early region 1A (E1A) proteins were prepared from BALB/c mice immunized with a bacterial trpE-E1A fusion protein. This protein is encoded by a hybrid gene that joins a portion of the Escherichia coli trpE gene and a cDNA copy of the E1A 13S mRNA (Spindler et al., J. Virol. 49:132-141, 1984). Eighty-three hybridomas that secrete antibodies which recognize the immunogen were isolated and single cell cloned. Twenty-nine of these antibodies are specific for the E1A portion of the fusion protein. Only 12 of the monoclonal antibodies can efficiently immunoprecipitate E1A polypeptides from detergent lysates of infected cells. E1A polypeptides were analyzed on one-dimensional, sodium dodecyl sulfate-polyacrylamide gels and two-dimensional, isoelectric focusing polyacrylamide gels. The E1A proteins that are specifically immunoprecipitated by the monoclonal antibodies are heterogeneous in size and charge and can be resolved into approximately 60 polypeptide species. This heterogeneity is due not only to synthesis from multiple E1A mRNAs, but also at least in part to post-translational modification. Several of the monoclonal antibodies divide the E1A polypeptides into immunological subclasses based on the ability of the antibodies to bind to the antigen. In particular, two of the monoclonal antibodies bind to the polypeptides synthesized from the 13S E1A mRNA, but not to other E1A proteins.     

The glycoprotein products of varicella-zoster virus gene 14 and their defective accumulation in a vaccine strain (Oka).

Many characteristics of the putative protein encoded by varicella-zoster virus (VZV) open reading fram (ORF) 14 indicate that it is a glycoprotein, which has been designated gpV. To identify the protein products of the gene, the coding sequences were placed under the control of the vaccinia virus p7.5 promoter and recombinant vaccinia viruses were constructed. Heterogeneous polypeptides with molecular weights of 95,000 to 105,000 (95K to 105K polypeptides) were expressed in cells infected by a vaccinia virus recombinant (vKIP5) containing ORF 14 from VZV Scott but were not expressed by control vaccinia viruses. These polypeptides were recognized by antibodies present in human sera that contained high levels of anti-VZV antibodies. Conversely, antisera raised in rabbits inoculated with vKIP5 reacted specifically with heterogeneous 95K to 105K polypeptides present in VZV Scott-infected but not uninfected cells; these polypeptides show a patchy plasma membrane fluorescence pattern in VZV Scott-infected cells. These same antisera neutralized VZV strain Scott infectivity in the absence of complement. Endoglycosidase F treatment of isolated gpV polypeptides and tunicamycin treatment of cells infected with the vKIP5 recombinant indicated that the polypeptides were glycosylated. Three sets of data imply that the VZV strain Oka, which has been used to produce a live attenuated virus vaccine, accumulates low levels of gpV polypeptides relative to wild-type strains: (i) blocking of antibodies in human sera with excess VZV Oka-infected cell antigen yielded residual antibodies which were reactive with the 95K to 105K gpV polypeptides expressed in cells infected by VZV strain Scott and by the vKIP5 vaccinia virus recombinant, but not with Oka-infected cell polypeptides; (ii) antisera raised to vKIP5 detected very low levels of reactive polypeptides made in VZV Oka-infected cells and neutralized VZV Oka virus much less efficiently than VZV Scott; and (iii) comparisons of the reactivity of sera from live attenuated virus vaccine vaccinees with sera derived from patients recovering from wild-type infections indicated greatly reduced levels of gpV-specific antibodies in some vaccinees.     

Immunochemical studies of DNA polymerase delta: relationships with DNA polymerase alpha

A panel of murine hybridoma cell lines which produce antibodies against polypeptides present in human placental DNA polymerase delta preparations was developed. Eight of these antibodies were characterized by virtue of their ability to inhibit DNA polymerase delta activity and immunoblot the 170-kDa catalytic polypeptide. Six of these eight antibodies inhibit DNA polymerase delta but not DNA polymerase alpha, showing that the two proteins are distinct. However, the other two monoclonal antibodies inhibited both DNA polymerase delta and alpha activities, providing the first evidence that these two proteins have a structural relationship. In addition to antibodies against the catalytic polypeptide we also identified 11 antibodies which recognize 120-, 100-, 88-, 75-, 62-, 36-, and 22-kDa polypeptides in DNA polymerase delta preparations, suggesting that these proteins might be part of a replication complex. The antibody to the 36-kDa polypeptide was shown to be directed against proliferating cell nuclear antigen/cyclin. These antibodies should prove useful for studies aimed at distinguishing between DNA polymerases alpha and delta and for the investigation of the functional roles of DNA polymerase delta polypeptides.     

Immunoblotting of keratin polypeptides extracted from tissues preserved in standard histologic fixatives

Cytoskeletal polypeptides from fresh placental tissue, tissue stored at -30 degrees C, and tissue fixed in 10% buffered formalin, Bouin's solution, and Carnoy's solution were extracted, separated by electrophoresis, and immunoblotted using monoclonal antibodies immunoreactive with keratin polypeptides. Storage of the placental tissue at -30 degrees C, or fixation in Carnoy's solution did not alter the extractability, migration pattern, or immunoreactivity of the keratin polypeptides. Keratin polypeptides could not be adequately demonstrated in extracts prepared from formalin- or Bouin's solution-fixed tissues. Several unmasking procedures used on tissues before extraction and on nitrocellulose blots before application of primary antibodies failed to unmask keratin polypeptides, either in Coomassie blue-stained gels or in immunoblots reacted with anti-keratin antibodies. These data indicate that Carnoy's solution is the fixative of choice for tissues in which electrophoretic and immunoblotting analyses of keratin polypeptides might be required.     

Monoclonal antibodies specific for the light-harvesting chlorophyll a/b-protein complex (LHC): Detection of conserved antigenic determinants in LHC from different species

Monoclonal antibodies have been raised against the light-harvesting chlorophyll a/b-protein complex (LHC) of pea and characterised using the enzyme linked immunosorbent assay (with purified LHC or intact thylokoids) or immunoblotting using chloroplast proteins transferred from SDS-PAGE gels. Several clones showed strong binding to the two major polypeptides of pea LHC, even after trypsin or proteinase K treatment. The two antibodies with the most efficient binding to pea LHC were shown to cross-react with polypeptides from green algae and higher plants; indicating sequential similarities and the presence of several closely related polypeptides between phylogenetically distant species.     

Application of denatured, electrophoretically separated, and immobilized lysates of herpes simplex virus-infected cells for detection of monoclonal antibodies and for studies of the properties of viral proteins

We report the use of herpes simplex virus type 1 (HSV-1)- and HSV-2-infected cell polypeptides (ICPs) separated by electrophoresis in polyacrylamide gels and transferred to nitrocellulose to (i) detect monoclonal antibodies to viral polypeptides and to (ii) study the properties of the proteins with the monoclonal antibodies. Our results were as follows. (i) When the antigens were electrophoretically separated in denaturing gels and then immobilized on nitrocellulose strips, we detected a greater diversity of monoclonal antibodies to viral proteins than when we used the technique of immune precipitation of soluble, nondenatured viral antigens. The primary advantage of the technique is in the detection of nonprecipitating antibody and of antibody to poorly soluble antigens not available for reaction in preparations cleared by high-speed centrifugation before immune reaction. (ii) Studies of the viral polypeptides reactive with three monoclonal antibodies indicated that the technique can be used to investigate several properties of the antigens. Specifically, monoclonal antibody to ICP 4 confirmed the accumulation of viral protein in the nucleus and the mapping of the gene in the S component. The results showed, however, that HSV-1 and HSV-2 ICP 4 do have common antigenic determinants. The reaction of a nonprecipitating monoclonal antibody with electrophoretically separated, immobilized polypeptides contained in cytoplasmic and nuclear fractions, those chemically deglycosylated, or those specified by specific HSV-1 x HSV-2 intertypic recombinants identified the antigens reactive with the second monoclonal antibody as various forms of glycoprotein gC. Of particular interest was a set of four antigens, 39,000 to 46,500 in apparent molecular weight, reactive with each of several monoclonal antibodies. These studies showed that two polypeptides partition in the cytoplasm and two in the nucleus and that all comap with the previously mapped ICPs 35 and 37 in the region of the genome defined by the viral thymidine kinase gene on the left and the glycoprotein gA/B gene on the right. Unlike ICP 4 and gC, the four polypeptides are linked by intermolecular bisulfide bonds, inasmuch as the polypeptides were not at the expected locations upon denaturation and electrophoresis in the absence of reducing agents.     

Production of homo- and hetero-dimeric isozymes from two aldehyde oxidase genes of Arabidopsis thaliana.

Polyclonal antibodies were raised against synthetic peptides or recombinant polypeptides encoded by Arabidopsis atAO-1 and atAO-2 cDNAs, which have sequences similar to maize and animal aldehyde oxidase (AO) cDNAs. Anti-atAO-1 antibodies recognized AOalpha and AObeta among the three isoforms, AOalpha, AObeta, and AOgamma, detected in Arabidopsis seedlings after native PAGE, while anti-atAO-2 antibodies reacted with AObeta and AOgamma. The polypeptide specifically recognized by each antibody was collected as the Protein-A/IgG/antigen complex. The 150- and 145-kDa polypeptides were purified by SDS-PAGE and digested with Achromobacter Protease I. From the amino acid sequences and molecular masses of the derivative peptides, it was revealed that the 150- and 145-kDa polypeptides were the products of atAO-1 and atAO-2, respectively. Molecular masses of the native forms of AOalpha, AObeta, and AOgamma were estimated as approximately 290-300 kDa. These results suggest that AOalpha and AOgamma are homodimers consisting of atAO-1 and atAO-2 products, respectively, and that AObeta is a heterodimer of the atAO-1 and atAO-2 products.