Masking of epitopes in tissue sections

Summary Antisera to chicken brain antigen (CBA) isolated by hydroxyapatite chromatography from 8 M urea extracts following repeated extractions with phosphate buffer selectively decorate neurofilaments (NF) in neuronal perikarya, dendrites and axons. The antisera also reacted with GFA protein, the astrocyte-specific intermediate filament protein, as indicated by the adsorption of NF immunoreactivity following passage of the antisera through columns prepared with purified GFA protein. Moreover, the antisera stained the polypeptides of the NF triplet (70 kd, 150 kd, 200 kd) and GFA protein by the immunoblotting procedure. Monoclonal antibodies selectively decorating NF in tissue sections were isolated from a fusion of mouse myeloma cells with spleen cells of mice immunized with CBA. By the immunoblotting procedure the antibodies decorated the 150 kd NF polypeptide and GFA protein. No staining of glial filaments or any other structure on tissue sections was also observed with antibodies derived from another fusion strongly reacting with GFA protein on immunoblots. All antibodies (monoclonal and polyclonal) appeared to react with the same region of the GFA polypeptide as indicated by immunoblots of cleavage products.     

Analysis of the B-G antigens of the chicken MHC by two-dimensional gel electrophoresis

The B-G antigens of the chicken major histocompatibility complex (MHC) have been analyzed by high resolution two-dimensional (2-D) gel electrophoresis. Monoclonal antibodies recognizing a widely shared B-G determinant were used for immunoprecipitating the B-G antigens from radioiodinated, detergent-solubilized erythrocyte membrane preparations. The B-G antigens produce a variety of patterns on 2-D gels. The number of polypeptides within a B-G pattern varies among haplotypes from single polypeptide arrays showing slight microheterogeneity to complex patterns which contain as many as four or five polypeptide arrays differing in relative mobility and isoelectric point. Many of the patterns, but not all, include a polypeptide of Mr =48 kd focusing near pH 6.9. At present it is not understood whether the multiple polypeptides within some B-G patterns represent the expression of multiple B-G genes or whether they are the result of modifications of single gene products during biosynthetic processing. 2-D gel analyses were also used to confirm the assignment of the same B-G haplotype in several different inbred flocks and the fate of the B-G antigens in two B system recombinant haplotypes. The 2-D gel patterns of these highly polymorphic antigens provide evidence for a complexity of the B-G locus not previously demonstrated. This technique may serve to define more objectively the diverse chicken MHC haplotypes which are now recognized and characterized only by serological techniques using alloantisera and monoclonal antibodies with varying cross-reactivities.     

Isolation and chemical characterization of a melanoma-associated proteoglycan antigen

Many melanoma-associated antigens have been identified by monoclonal antibodies. One of these monoclonal antibodies, O₁-94-45, binds only to melanomas, nevus cells, some astrocytomas, and fetal epitheloid cells. There are approximately 100,000 cell surface antigens per melanoma cell with an association constant of 3 × 10⁸m^?1. The antigen is efficiently extracted from the membrane only in the presence of detergent and is, therefore, bound by hydrophobic forces. However, it is also shed into the culture supernatant during normal cell growth. The two components of the O₁-95-45 antigen are a chondroitin sulfate proteoglycan (CSP, >500,000 Da) and a glycoprotein gp260 (260,000 Da, pI 6.9). CSP contains chondroitin sulfate and N-linked and O-linked oligosaccharides. Only N-linked saccharides were associated with gp260. The antigenic site is expressed on both components and is heat-sensitive. Since the CSP was converted to gp260 by chondroitinase, the protein cores of the two molecules are the same or similar. For more detailed study the O₁-95-45 antigen was purified by immunoaffinity chromatography. The amino acid composition of the purified antigen was relatively polar with an unusually high Leu content and low Lys content. Initial attempts to sequence the antigen were unsuccessful probably due to a blocked N-terminus. CSP and gp260 were partially separated by gel filtration chromatography, and both were found to carry the O₁-95-45 antigenic determinant. Three other monoclonal antibodies were found to bind the purified antigen at a site or sites different from the O₁-95-45 epitope and one other monoclonal antibody may bind at the same site. Two of these antibodies were used for a double determinant immunoassay.     

Association between allo-immunoreactive and xeno-immunoreactive subunits of a glycoprotein tumor-associated antigen

Summary Using allogeneic antibody, we previously described a tumor-associated antigen (TAA) in the urine of 68% of melanoma patients. The TAA was purified from urine of a melanoma patient and used as immunogen to develop a murine monoclonal antibody (AD1-40F4) and xenopolyclonal antibodies in a baboon. Sera from melanoma patients treated with whole melanoma cell vaccine were used as the source of human antibody to the glycoprotein antigen. Treatment with 2-mercaptoethanol and separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis resolved the high-molecular-mass glycoprotein TAA into smaller subunits. Immunoblot analysis indicates that the murine monoclonal antibody (AD1-40F4) recognized a 90–100-kDa subunit of the antigen while human anti-TAA antibodies primarily recognized a 65-kDa subunit in addition to the 90–100-kDa subunit. Baboon polyclonal antibodies recognized the same subunits plus a 120-kDa subunit. Blocking studies indicated that the murine monoclonal and baboon polyclonal antibodies recognized the closely related epitopes on the 90–100-kDa subunit, while human antibodies recognized an epitope entirely distinct from that recognized by the mouse antibody. These results demonstrate the epitope complexity associated with the high-molecular-mass glycoprotein TAA.     

Monoclonal antibodies to human plasma Protein X alias complement S-protein

Protein X alias complement S-protein was isolated by dissociation from purified XCSb-9 (fluid-phase terminal C5b-9) complexes with 250 mM deoxycholate and subsequent sucrose density gradient centrifugation and Sephacryl gel chromatography. Polyclonal rabbit and monoclonal mouse antibodies were used to preliminarily characterize the protein in human serum and plasma. In plasma, Protein X yielded a symmetrical immuno-precipitate of ₂-mobility in a crossed immunoelectrophoresis assay. However, a second immunoprecipitate of Oh-mobility was observed when serum was analysed; this precipitate represented Protein X in complex with antithrombin-III. The co-precipitation of Protein X with serum antithrombin-III was exploited for establishing a simple screening test for unequivocal identification of monocJonal anti - Protein X antibodies. SDS-PAGE immunoblotting with monoclonal antibodies showed that Protein X exhibits pronounced microheterogeneity, migrating as a diffuse moiety of approx. M r 80-90 000. Additionally, a small amount of polymeric aggregates appear to be present in plasma. Reduction of disulfide bonds led to liberation of a polypeptide of approx. 15 K as discerned by two-dimensional SDS-PAGE immunoblotting. Protein X is not cleaved to lower molecular weight entities during the process of blood coagulation or during formation of fluid-phase terminal complement complexes. The plasma concentrations in healthy adults were in the range of500–700 pg/ml. The availability of methods for isolating Protein X and raising monoclonal antibodies will facilitate further studies on the dual role of this protein in the terminal complement and coagulation cascades.     

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

Abstract. A panel of 10 mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFA) has been isolated using porcine GFA as antigen. Although all antibodies recognize GFA purified from porcine spinal cord in the western blot technique, they can be subdivided into at least three groups on the basis of their reactivity against defined fragments of the molecule. Immunofluorescence staining patterns with the monoclonal antibodies performed on tissues and cell lines resemble those reported with conventional polyclonal antibodies directed against GFA. In particular astrocytes and Bergmann glia are strongly stained. In addition mouse monoclonal antibodies specific for either the 200 kd, or the 160 kd, or the 68 kd neurofilament triplet protein have been isolated and characterized. These antibodies are specific for neuronal cells and support conclusions made with similar antigen affinity-purified polyclonal antibodies. The combined set of monoclonal antibodies seems a valuable tool to characterize the different cell types of the nervous system.     

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

A panel of 10 mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFA) has been isolated using porcine GFA as antigen. Although all antibodies recognize GFA purified from porcine spinal cord in the western blot technique, they can be subdivided into at least three groups on the basis of their reactivity against defined fragments of the molecule. Immunofluorescence staining patterns with the monoclonal antibodies performed on tissues and cell lines resemble those reported with conventional polyclonal antibodies directed against GFA. In particular astrocytes and Bergmann glia are strongly stained. In addition mouse monoclonal antibodies specific for either the 200 kd, or the 160 kd, or the 68 kd neurofilament triplet protein have been isolated and characterized. These antibodies are specific for neuronal cells and support conclusions made with similar antigen affinity-purified polyclonal antibodies. The combined set of monoclonal antibodies seems a valuable tool to characterize the different cell types of the nervous system.     

Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine

Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn²⁺-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.Abbreviations GS glutamine synthetase - MOPS 2-(N-morpholine) propane sulfonate - MSX l-methionine-d,l-sulfoximine     

Estradiol receptor: phosphorylation on tyrosine in uterus and interaction with anti-phosphotyrosine antibody.

Estradiol receptor from rat uteri incubated with [32P] orthophosphate has been purified by diethylstilbestrol--Sepharose followed by heparin--Sepharose chromatography. The purified receptor, analyzed by centrifugation through sucrose gradients after incubation with monoclonal antibodies against purified estradiol receptor, appears to be labeled with 32P. The receptor preparation has been further purified by immunoaffinity chromatography and submitted to SDS--poly-acrylamide gel electrophoresis. A heavily 32P-labeled 68 kd protein and a very lightly 32P-labeled 48 kd protein, probably a proteolytic product of the 68 kd protein, were detected. Phosphoamino acid analysis of the receptor eluted from the immunoaffinity column shows that its 32P-labeling occurs exclusively on tyrosine. This is the first report on phosphorylation on tyrosine of a steroid receptor in tissue. It is consistent with our previous finding that a uterus estradiol receptor-kinase, which confers hormone binding ability to the estradiol receptor, in vitro phosphorylates this receptor exclusively on tyrosine. Calf uterus receptor binds with high specificity and affinity to monoclonal anti-phosphotyrosine antibodies covalently bound to Sepharose (Kd = 0.28 nM). Dephosphorylation of the receptor by nuclei containing the calf uterus nuclear phosphatase abolishes the interaction with antibodies. These results suggest that also in calf uterus, estradiol receptor is phosphorylated on tyrosine. Anti-phosphotyrosine antibodies bound to Sepharose have been used to partially purify the estradiol receptor from calf uterus.     

Glycoprotein (gp-51) antigen not associated with the bovine leukemia virus in FLK culture supernatant liquid]

After elimination of the virus fraction non-associated with bovine leukemia virus (free) glycoprotein gp-51 antigen has been preparatively isolated from the supernatant of the FLK cell culture. For this purpose ultrafiltration through PM-30 membrane, thrice-repeated isoelectric focusing with different (pH 3.0-10.0, pH 4.0-6.0) ampholine intervals and affinity chromatography with ConA-sepharose are used. The SDS-polyacrylamide gel electrophoresis has determined it to be a homogeneous protein with molecular weight 51,000 daltons. The results of the enzyme-linked immunosorbent assay, immunoelectrophoresis and agar-gel immunodiffusion test confirm the specific activity of isolated gp-51 antigen. The gp-51 antigen may be used for identification of antibodies in the blood serum of leukemia virus of infected cattle and for monoclonal screening of antibodies.     

Purification of the major UsnRNPs from broad bean nuclear extracts and characterization of their protein constituents.

Small nuclear ribonucleoprotein particles containing the five major nucleoplasmic snRNAs U1, U2, U4, U5 and U6 as well as two smaller sized snRNAs were purified from broad bean nuclear extracts by anti-m3G, monoclonal antibody, immunoaffinity chromatography. We have so far defined 13 polypeptides of approximate mol. wts. of 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 18.5 kd, 25 kd (double band), 30 kd, 31 kd, 35 kd, 36 kd and 54 kd. Upon fractionation of the UsnRNPs by anion exchange chromatography, essentially pure U5 snRNPs were obtained, containing the 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 35 kd and 36 kd polypeptides. These may therefore represent the common snRNP polypeptides and which may also be present in the other snRNPs. By immunoblotting studies, using anti-Sm sera and mouse monoclonal antibodies we show that the 35 kd and 36 kd proteins are immunologically related to the mammalian common B/B' proteins. The broad bean 16 kd and 17 kd proteins appear to share structural elements with the mammalian D protein. The three proteins of mol. wts. 11 kd, 11.5 kd and 12.5 kd probably represent the broad bean polypeptides E, F, and G. Cross-reactivity of proteins of mol. wts of 30 kd and 31 kd with Anti-(U1/U2)RNP antibodies suggests that they may represent the broad bean A and B" polypeptides. The 54 kd protein and the 18.5 kd protein could be candidates for the U1 specific 70 k and C polypeptides. Our results demonstrate a strong similarity between the overall structure of broad bean and mammalian snRNPs.     

DNA-binding region of the simian virus 40 tumor antigen.

The simian virus 40 (SV40) tumor (T) antigen was purified by immunoaffinity chromatography and cleaved with small amounts of trypsin, and the resulting fragments were subjected to SV40 DNA cellulose chromatography. A 44,000-molecular-weight fragment (44K fragment) from the left end of the molecule and a 30K fragment mapping from approximately Lys 131 to Lys 371 bound to the column and were eluted with 1 M NaCl. In a second series of experiments, T antigen was immunoprecipitated with hamster anti-T serum or various monoclonal antibodies and partially digested with trypsin. Fragments that were solubilized by this treatment were tested for DNA-binding activity by using an SV40 DNA fragment-binding assay. A 17K fragment which originated from the amino-terminal region of the polypeptide had no apparent binding activity in this assay. On the other hand, larger fragments (76K, 46K, and 30K) whose amino termini were mapped around Lys 131 did display DNA-binding activity. Finally, complexes consisting of SV40 DNA and T-antigen fragments were precipitated in the DNA-binding assay with monoclonal antibodies that recognize the central region of the protein; however, antibodies with specificities to the amino- or carboxy-terminal regions were inactive. These results strongly suggest that the DNA-binding region of T antigen lies approximately between Lys 131 and Lys 371, corresponding to 0.51 and 0.37 map units on the DNA.     

Interrelationships of the “Ly-6 complex” antigens

Competitive binding studies and immunoprecipitation experiments define at least five distinct epitopes encoded by Ly-6-linked genes—Ly-6A.2, Ly-6B.2, Ly-6C.2, Ly-6D.2, and ThB. Ly-6A.2, a 33 kd protein, and Ly-6D.2 are closely overlapping epitopes that can be distinguished by their unique thymus reactions of 10–20% or >90%, respectively. Similarly, the Ly-6C.2 antigen present on a 14 kd moiety loosely overlaps the Ly-6B.2 antigen. Ly-6C.2 and Ly-6B,2 antigens are distinct from Ly-6A.2 and Ly-6B.2, however. ThB is a 16–18 kd antigen which is not associated on the cell surface with any other Ly-6 antigens. In addition, independently derived antibodies made to the Ly-6C.2 antigen detect an identical epitope, as do antibodies to Ly-6A.2 and Ly-6B.2. These results imply the existence of a single antigenic site on each of these molecules.     

Purification and characterisation of MHC class I antigen from rat liver with monoclonal antibody

We have described three monoclonal antibodies (HAM1, HAM2, and HAM3) to rat liver cell membrane glycoproteins. Recently also we reported another monoclonal antibody (HAM4) to rat hepato-renal membrane antigen. Using these monoclonal antibodies, it is possible to purify membrane antigens. This paper describes the details of the purification and the nature of the antigen purified with one of the monoclonal antibodies (HAM2) to rat liver cell membrane glycoproteins. Antigen was purified with immunoaffinity column. The amino acid composition was determined and compared with those of mice MHC class I antigen (H-2) and with the rat lymphocyte membrane antigens which were purified with monoclonal antibodies and of which amino acids compositions were determined.     

Identification of the components of the murine T cell antigen receptor complex

In addition to the alpha and beta chains of the MHC class II restricted antigen receptor, monoclonal anti-receptor antibodies coprecipitate four polypeptides that appear to be noncovalently associated with the alpha-beta dimer of murine T cells. Included in the murine T cell antigen receptor complex are two glycoproteins of 25 kd (gamma) and 21 kd (delta) and two nonglycosylated polypeptides of 26 kd (epsilon) and 16 kd (zeta). The epsilon chain appears to possess an intrachain disulfide bond and zeta exists in the complex as a disulfide-linked homodimer. The delta chain is phosphorylated on a serine residue in response to T cell activation with antigen. In contrast, both delta and epsilon are phosphorylated in response to treatment of the T cells with phorbol 12-myristate 13-acetate. These polypeptides may play a role in the transduction of the signal(s) in T cell activation.     

Purification and characterization of the D2 cell adhesion protein: Analysis of the postnatally regulated polymorphic forms and their cellular distribution

The developmentally regulated, D2 cell adhesion protein has been purified from 10–12 day old rat synaptosomes by sequential hydroxyapatite chromatography, wheat germ lectin affinity chromatography and gel filtration. The purified protein was found to be composed of two polypeptide components of 200 and 140 kd molecular weight which comprised 0.5–1.0% of total synaptosomal membrane protein. Lysine-Sepharose affinity chromatography could further separate the purified protein into sialic acid-rich and sialic acid-poor forms. Immunoblot analysis of whole brain homogenates and synaptosomes with an antiserum raised against the purified protein (anti-D2) revealed the presence of three immunologically related polypeptides of 200, 140, and 115 kd molecular weight. These polypeptides, which appeared as a diffuse zone (>200 kd) in fetal material, were found to developmentally regulate by altering their relative expression. This was particularly marked in the 200 kd component. Furthermore, the 200 kd polypeptide appeared to be neuron-specific as both the 140 and 115 kd components were common to synaptosomes and primary cultures of astrocytes.     

Evaluation of the antigen from chlamydospores of Candida albicans in the serodiagnosis of candidiasis

Antigens have been prepared from the chlamydospores and blastospores of Candida albicans and their precipitin patterns were analysed by two-dimensional immunoelectrophoresis using specific antisera.The two antigens were used in routine serological tests of patients suffering from candidiasis. On double-diffusion tests for the detection of circulating antibodies of Candida albicans, the antigen from chlamydospores displays precipitin lines that differ in number and intensity from those obtained with the antigen from blastospores. The results are briefly discussed in the framework of C. albicans antigen standardization.     

Characterization of the antigen identified by Po66

Summary The mouse monoclonal antibody (mAb) Po66 has been shown in previous work to be localized in nude mice xenografts of human lung tumours when injected intravenously [Dazord L et al. (1987) Cancer Immunol Immunother 24: 263–268] and to be suitable for the scintigraphic detection of lung cancers in patients [Dazord L, et al. (1987) in Klapdor (ed) New tumour markers and their monoclonal antibodies. Georg Thieme, Stuttgart, New York, pp 444–450]. The nature of the antigen recognized by Po66 has been investigated in the present work and comparisons are made with antigens recognized by other mAbs prepared in the laboratory. These mAbs were raised either against lung squamous cell carcinoma (mAbs Po43, Po60), or against a bronchio-alveolar carcinoma (mAbs BAM33, BAM45, BAM54 and BAM69). Radioiodinated purified Po66 did not compete for cell binding with any other mAb. All Po and BAM mAbs reacted with tumour cells both cultured in vitro and grown in vivo. They recognized cytoplasmic antigens as judged by immunofluorescence examination of fixed cells or by immunoperoxidase staining of cancer tissues, but could never be visualized by immunofluorescence on the surface membrane of culture cells. The mAbs of the BAM series reacted with vimentin as demonstrated by immunofluorescence staining, showing alterations in the aspect of the filaments under the effect of colchicin. Radiolabelled mAbs Po43, BAM33 and BAM45 bound to partially purified cytoplasmic cytoskeleton components. In contrast, Po66 was never seen associated with intermediary filaments. The sensitivity to enzyme digestion of the antigen associated with Po66 was studied in comparison with those associated with Po43, BAM33 and BAM45. All antigens were sensitive to protease digestion while only the Po66-identified antigen was sensitive to periodate, neuraminidase and -fucosidase. Thus, mAb Po66 identified an antigen of 47 kDa (as determined before) present in the cytoplasm but not related to the cytoskeleton, not detected on the cell surface and glycoproteic in nature.     

DNA helicase activity of SV40 large tumor antigen.

Large tumor antigen (T antigen) was extracted from SV40-infected African Green Monkey cells and purified to homogeneity by immunoaffinity chromatography. The purified T antigen preparations unwind DNA duplices of greater than 120 bp in a reaction which is dependent on magnesium ions and ATP hydrolysis. Based on these and other properties of the reaction we classify this newly discovered enzymatic activity as a eukaryotic DNA helicase. The helicase and the known ATPase function of T antigen cosediment with the mono- or dimeric 4-6 S form of T antigen, but not with higher T antigen aggregates. The helicase activity seems to be an intrinsic function of SV40 T antigen. First, several different T antigen-specific monoclonal antibodies interfere with the DNA unwinding activity; monoclonals which are known to reduce the T antigen-specific ATPase most strongly inhibited the helicase reaction. Second, mutant T antigens with impaired ATPase function also showed a reduced DNA unwinding activity.     

小鼠组织相容性抗原的提取与纯化

以建立方便、大量纯化组织相容性抗原的方法为目的。用０．５％Ｔｒｉｔｏｎ／Ｔｒｉｓ抽提小鼠组织相容性抗原（Ｈ－２）抗原,利用抗Ｈ－２抗原抗体制备的亲和柱,特异性结合Ｈ－２抗原,再用０．５％ＤＯＣ、０．６５ＭＮａＣｌ洗脱结合Ｈ－２抗原。结果显示：电泳显示纯化物为４５ｋｄ（重链）,１２ｋｄ（轻链）两条带,纯化物具有明显的血清学及生物学活性;这种亲和层析法可大量纯化组织相容性抗原,用于器官移植研究及组织相容性抗原的免疫功能研究。