Comodulation of CD3 and CD4. Evidence for a specific association between CD4 and approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes

The aggregation of a specific class of lymphocyte surface molecules results in patching, capping, and surface modulation of the aggregated ligand. Both CD4, an associative recognition structure found on helper T lymphocytes, and CD3, a component of the T cell receptor complex, are members of this functional subgroup. When 125I-labeled monoclonal antibodies reactive with either CD4 (19Thy 5D7) or CD3 (RW24B6) were bound to T lymphocytes, the subsequent addition of goat anti-mouse Ig resulted in their rapid, temperature-dependent internalization. Whereas the binding of 125I-19Thy 5D7 (anti-CD4) was inhibited by greater than 90% in the presence of unlabeled 19Thy 5D7, no inhibition occurred in the presence of unlabeled antibody reactive with CD3 (RW28C8). We took advantage of the fact that these antibodies were of different isotypes (19Thy 5D7:IgG2a; RW28C8:IgGl) to determine whether the internalization of CD3 induced the comodulation of CD4. T lymphocytes preincubated with 125I-19Thy5D7 (anti-CD4) and unlabeled RA28C8 (anti-CD3) were treated with goat anti-mouse IgGl under conditions shown to quantitatively internalize CD3. After 1 h at 37 degrees C, T lymphocytes had internalized 10.5 +/- 2.6% (n = 3) of their antibody-bound cell surface CD4. After similar incubations with media alone or with goat anti-mouse IgGl in the absence of prebound RW28C8 (anti-CD3), no internalization of CD4 could be detected. Control antibodies reactive with CD45R (2H4, IgGl) also failed to induce the internalization of CD4. Similar results were obtained by using a helper T cell clone (T4C1) that internalized 9.6 +/- 2.8% (n = 3) of its antibody-bound cell surface CD4 in response to CD3 modulation. In a reciprocal experiment, 125I-anti-CD3 (RW24B6, IgG2b) was preincubated with T4Cl cells together with unlabeled anti-CD4 (12T4D11, IgG1) prior to the addition of goat anti-mouse IgGl. The quantitative modulation of CD4 induced the co-internalization of 4.6 +/- 0.6% (n = 3) of cell surface CD3. These results suggest that approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes are specifically associated with CD4. Furthermore, our results suggest that an average of two CD4 molecules associate with each CD3:T cell receptor complex.     

The murine IL 2 receptor. I. Monoclonal antibodies that define distinct functional epitopes on activated T cells and react with activated B cells

The properties of three distinct rat monoclonal antibodies, designated 3C7, 7D4, and 2E4, to the murine IL 2 receptor have been compared in binding, biochemical, and functional assays. 3C7 appears to define an epitope near or identical to the IL 2-binding site of the receptor, because 3C7 inhibited the binding of radiolabeled IL 2 to CTL-L cells and because unlabeled IL 2 inhibited the binding of FITC-3C7 to CTL-L cells. 7D4 and 2E4 had no effect on IL 2 binding. Competitive antibody-binding studies confirmed that the epitope seen by 3C7 was distinct from the epitope(s) seen by 7D4 and 2E4. Sequential immunoprecipitation studies demonstrated that all three antibodies were reactive with the same molecular species, and that each precipitated identical components of 20,000 to 25,000 daltons, 50,000 to 60,000 daltons, and 100,000 to 120,000 daltons from the surface of CTL-L. FACS studies demonstrated a quantitatively and qualitatively identical cell distribution for the antigen defined by each antibody. They failed to stain more than 95% of resting lymphocytes, but were strongly reactive with Con A T blasts and substantially less reactive with LPS B blasts. Unlabeled IL 2 was also able to inhibit the binding of FITC-3C7 to LPS B cell blasts, suggesting the presence of IL 2-binding sites on activated B cells. Each antibody inhibited IL 2-driven proliferation of HT2 or CTL-L cells. 3C7 and 7D4 were more potent inhibitors of proliferation than was 2E4, and the combined use of 3C7 and 7D4 resulted in greater levels of inhibition of proliferation than that shown from the use of either antibody alone. Collectively, the results support the hypothesis that these antibodies detect two distinct functional regions of the IL 2 receptor.     

Antigenic Heterogeneity of Qa-2 Antigens in C57BL/6

The Qa-2 antigens are class I-like molecules encoded by genes mapped telomeric to the H-2D region on chromosome 17 in the mouse. A panel of 8 new monoclonal anti-Qa-2 antibodies derived from a C3H.KBR anti-C3H. SW immunization was studied. Immunoprecipitation of¹²⁵I-labeled C57BL/6 splenocyte antigens showed that all of these antibodies precipitated 40 kDa molecules which could be completely precleared by the monoclonal antibody 20-8-4, which had previously been shown to crossreact with Qa-2. One of the monoclonal antibodies (1-12-1), however, was found not to completely preclear Qa-2 antigens precipitable by the other 7 antibodies or by 20-8-4, suggesting the existence of at least two different species of Qa-2 molecules. Cell lines transfected with Q7 or Q9 genes were reactive with all 9 antibodies and the Qa-2 antigens expressed on surface membranes of these cells were completely precleared by both 20-8-4 and 1-12-1. Therefore, the observed heterogeneity of these molecules cannot be explained by an antigenic difference between the Q7 and Q9 gene products. 2D gel analyses showed identical pI spectra between Qa-2 molecules precipitated with 20-8-4 and 1-12-1. In addition, all of the monoclonal antibodies reacted with labeled antigen preparations following treatment with Endo F or neuraminidase, indicating that carbohydrate moieties are probably not responsible for the antigenic difference between the two species of Qa-2 antigen.     

Monoclonal antibody for calcitriol (1 alpha,25-dihydroxyvitamin D3)

Hybridoma cell lines secreting antibodies for vitamin D3 metabolites have been generated by fusing splenocytes from BALB/c mice immunized with 3 beta-glutaryl-25-hydroxyvitamin D3 conjugated to bovine serum albumin (3 beta-glu-25-OH-D3-BSA) and Sp2/O-Ag14 myeloma cells. Purification of monoclonal antibodies from culture media or ascites fluids was accomplished by procedures including affinity chromatography on Protein A-Sepharose 4B. Each monoclonal antibody was analyzed as to its affinity and specificity by equilibrium dialysis and an enzyme immunoassay (EIA) based on a double antibody system. It was demonstrated that clone 1C2-60 produced an antibody highly specific to 1 alpha,25-dihydroxyvitamin D3 (calcitriol), and the clone 2B3-66 antibody was reactive to 25-hydroxyvitamin D3 and similar structural compounds. These two monoclonal antibodies produced by 1C2-60 and 2B3-66 were determined to belong to the IgG2a class, and their affinity constants (Ka) with 3 beta-glu-25-OH-D3 were demonstrated to be 3.6 X 10(9) M-1 and 2.9 X 10(9) M-1, respectively, at 4 degrees C. The characteristics of these monoclonal antibodies were compared with those of conventional antibodies raised in mice and rabbits. Finally, by using monoclonal antibody 1C2-60, a sensitive EIA has been developed that can detect 10 pg of calcitriol.     

Chloroacetates of 2- and 3-demethylthiocolchicine: specific covalent interactions with tubulin with preferential labeling of the beta-subunit.

We synthesized two chemically reactive A ring modified analogs of colchicine, 2-chloroacetyl-2-demethylthiocolchicine (2-CTC) and 3-chloroacetyl-3-demethylthiocolchicine (3-CTC). Both are similar to colchicine as inhibitors of tubulin polymerization and act as competitive inhibitors of colchicine binding (apparent Ki values, 3 microM). [14C]-labeled 2-CTC and 3-CTC bound to tubulin at 37 degrees C but not at 0 degree C, and bound drug formed covalent bond(s) with tubulin. The binding and covalent reactions were inhibited by podophyllotoxin. About 60% of the bound 3-CTC rapidly formed a covalent bond with tubulin. With 2-CTC the covalent reaction was slower than the binding reaction, and only one-third of the bound 2-CTC reacted covalently with tubulin. The ratio of radiolabel in beta-tubulin to that in alpha-tubulin was about 4:1 with both 2-CTC and 3-CTC.     

Localization of binding sites for carboxyl terminal specific anti-rhodopsin monoclonal antibodies using synthetic peptides

The binding sites for four monoclonal antibodies, rho 1D4, rho 3C2, rho 3A6, and rho 1C5, have been localized within the C-terminal region of bovine rhodopsin: Asp18'-Glu-Ala16'-Ser-Thr-Thr-Val12'-Ser-Lys-Thr-Gl u8'-Thr-Ser-Gln-Val4'-Ala-Pr o -Ala1'. Antibody binding sites were localized by using synthetic C-terminal peptides in conjunction with solid-phase competitive inhibition assays and limited proteolytic digestion of rhodopsin in conjunction with electrophoretic immunoblotting techniques. Binding of the rho 1D4 and rho 3C2 antibodies to immobilized rhodopsin was inhibited with peptides of length 1'-8' and longer. Antibody rho 1D4 binding was not inhibited by peptides 2'-13' or 3'-18', indicating that the C-terminal alanine residue of rhodopsin was required. Similar competitive inhibition studies indicated that the antibody rho 3A6 required peptides of length 1'-12' and longer whereas rho 1C5 required peptide 1'-18'. Peptide 3'-18' was as effective as 1'-18' in inhibiting rho 3A6 binding to rhodopsin, but replacement of glutamic acid in position 8' with glutamine abolished competition. This substitution had little effect on the binding of antibody rho 1C5. Thus, Glu8' was essential for rho 3A6 binding but not for the binding of the rho 1C5 antibody. Cleavage of the seven amino acid C-terminus from rhodopsin and further cleavage to F1 (Mr 25 000) and F2 (Mr 12 000) fragments with Staphylococcus aureus V8 protease abolished binding of rho 1D4 antibody to the membrane-bound rhodopsin fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin 2 receptors on cultured murine epidermal Langerhans cells

Rat monoclonal antibodies 3C7 and 7D4 detect two distinct functional regions of the murine interleukin 2 (IL 2) receptor. When studying the emergence kinetics of IL 2 receptors in mixed epidermal cell (EC)-lymphocyte cultures by using 3C7 and 7D4 in an indirect immunofluorescence assay, we regularly encountered a distinctive membrane fluorescence not only on lymphocytes, but also on a subpopulation of cells exhibiting a dendritic morphology. Reasoning that these 3C7/7D4-reactive dendritic cells might represent a subpopulation of epidermal dendritic cells, we studied mouse EC for the presence of 3C7/7D4- reactive cells. Although 3C7/7D4 reactivity was never detected on freshly isolated EC or on epidermal sheets, a small number of 3C7/7D4+ cells was encountered after 24 to 48 hr of culture. These cells exhibited a dendritic shape, expressed Ia antigens, lacked Thy-1 antigens, and displayed the ultrastructural features of Langerhans cells (LC) with the notable exception of Birbeck granules. Although after 24 hr, only 20% of Ia+ EC were 3C7/7D4+, the vast majority of LC displayed 3C7/7D4 binding sites after 4 to 5 days of culture. Preincubation of cultured LC-enriched EC with recombinant human IL 2 prevented subsequent 3C7-but not 7D4-binding to these cells. Western blot analysis of 7D4-reactive material of detergent extracts from LC-enriched EC revealed three bands in the same m.w. range as reported for CTLL cells. These results demonstrate that cultured LC express IL 2 receptors and may bear important implications for a better understanding of growth regulation, differentiation, and immunologic functions of LC.     

Photoaffinity approaches to determining the sequence selectivities of DNA-small molecule interactions: actinomycin D and ethidium.

The DNA photoaffinity ligands, 7-azidoactinomycin D and 8-azidoethidium, form DNA adducts that cause chain cleavage upon treatment with piperidine. Chemical DNA sequencing techniques were used to detect covalent binding. The relative preferences for modifications of all possible sites defined by a base pair step (e.g. GC) were determined within all quartet contexts such as (IGCJ). These preferences are described in terms of 'effective site occupations', which express the ability of a ligand to covalently modify some base in the binding site. Ideally, the effective site occupations measured for photoaffinity agents can also be related to site-specific, non-covalent association constants of the ligand. The sites most reactive with 7-azidoactinomycin D were those preferred for non-covalent binding of unsubstituted actinomycin D. GC sites were most reactive, but next-nearest neighbors exerted significant influences on reactivity. GC sites in 5'-(pyrimidine)GC(purine)-3' contexts, particularly TGCA, were most reactive, while reactivity was strongly suppressed for GC sites with a 5'-flanking G, or a 3'-flanking C. High reactivities were also observed for bases in the first (5') GG steps in TGGT, TGGG and TGGGT sequences recently shown to bind actinomycin D with high affinity. Pyrimidine-3',5'-purine steps and GG steps flanked by a T were most preferred by 8-azidoethidium, in agreement with the behavior of unsubstituted ethidium. The good correspondence between expected and observed covalent binding preferences of these two azide analogs demonstrates that photoaffinity labeling can identify highly preferred sites of non-covalent DNA binding by small molecules.     

Studies of Atlantic salmon (Salmo salar L.) blood, spleen and head kidney leucocytes using specific monoclonal antibodies, immunohistochemistry and flow cytometry

Monoclonal antibodies (mabs) raised against Atlantic salmon serum IgM (C7G7 and G2H3) and isolated peripheral blood leucocytes (PBL) (E3D9, C4B6 and D8B3) were applied in this study. Using immunoenzymehistochemistry, immunofluorescence and flow cytometry, the distribution of mab+ cells in blood, spleen and head kidney from Atlantic salmon were studied. Immunostaining on cytospin preparations and flow cytometry of isolated PBL showed that the Ig+ cells recognised by C7G7 and G2H3 were mononuclear leucocytes (MNL). The cytospin preparations showed some Ig+ cells with strong cytoplasmic staining, most likely plasma cells. The salmon blood neutrophils were the only E3D9+ cells in cytospin preparations of PBL, and E3D9 recognised about 94% of the defined neutrophil fraction in flow cytometry. The reactivities of C4B6 and D8B3 were to a large degree similar in both immunoenzymehistochemistry and flow cytometry, recognising both MNL and blood neutrophils. Immunofluorescence double staining of PBL with C4B6 and D8B3 showed double staining of all mab+ cells and D8B3 was apparently not able to block the binding of C4B6 to PBL. Immunofluorescence double staining of PBL also revealed more E3D9+ than C4B6+ neutrophils. In immunostaining on cryostat sections of spleen and head kidney, staining of cells was observed with all the mabs, the head kidney generally containing more positive cells than the spleen. Some potential applications for immunological studies using these mabs are suggested.     

Studies of Atlantic salmon (Salmo salar L.) blood, spleen and head kidney leucocytes using specific monoclonal antibodies, immunohistochemistry and flow cytometry

Monoclonal antibodies (mabs) raised against Atlantic salmon serum IgM (C7G7 and G2H3) and isolated peripheral blood leucocytes (PBL) (E3D9, C4B6 and D8B3) were applied in this study. Using immunoenzymehistochemistry, immunofluorescence and flow cytometry, the distribution of mab+ cells in blood, spleen and head kidney from Atlantic salmon were studied. Immunostaining on cytospin preparations and flow cytometry of isolated PBL showed that the Ig+ cells recognised by C7G7 and G2H3 were mononuclear leucocytes (MNL). The cytospin preparations showed some Ig+ cells with strong cytoplasmic staining, most likely plasma cells. The salmon blood neutrophils were the only E3D9+ cells in cytospin preparations of PBL, and E3D9 recognised about 94% of the defined neutrophil fraction in flow cytometry. The reactivities of C4B6 and D8B3 were to a large degree similar in both immunoenzymehistochemistry and flow cytometry, recognising both MNL and blood neutrophils. Immunofluorescence double staining of PBL with C4B6 and D8B3 showed double staining of all mab+ cells and D8B3 was apparently not able to block the binding of C4B6 to PBL. Immunofluorescence double staining of PBL also revealed more E3D9+ than C4B6+ neutrophils. In immunostaining on cryostat sections of spleen and head kidney, staining of cells was observed with all the mabs, the head kidney generally containing more positive cells than the spleen. Some potential applications for immunological studies using these mabs are suggested.     

Monoclonal Antibodies Specific for the Different Subunits of Asymmetric Acetylcholinesterase from Chick Muscle

The asymmetric (20S) acetylcholinesterase (AChE, EC 3.1.1.7) from 1-day-old chick muscle, purified on a column on which was immobilised a monoclonal antibody (mAb) to chick brain AChE, was used to immunise mice. Eight mAbs against the muscle enzyme were hence isolated and characterised. Five antibodies (4A8, 1C1, 10B7, 7G8, and 8H11) recognise a 110-kilodalton (kDa) subunit with AChE catalytic activity, one antibody (7D11) recognises a 72-kDa subunit with pseudocholinesterase or butyrylcholinesterase (BuChE, EC 3.1.1.8) catalytic activity, and two antibodies (6B6 and 7D7) react with the 58-kDa collagenous tail unit. Those three polypeptides can be recognised together in the 20S enzyme used, which is a hybrid AChE/BuChE oligomer. Antibodies 6B6 and 7D7 are specific for asymmetric AChE. Four of the mAbs recognising the 110-kDa subunit were reactive with it in immunoblots. Sucrose density gradient analysis of the antibody-enzyme complexes showed that the anti-110-kDa subunit mAbs cross-link multiple 20S AChE molecules to form large aggregates. In contrast, there is only a 2-3S increase in the sedimentation constant with the mAbs specific for the 72-kDa or for the 58-kDa subunit, suggesting that those subunits are more inaccessible in the structure to intermolecular cross-linking. The 4A8, 10B7, 7D11, and 7D7 mAbs showed cross-reactivity to the corresponding enzyme from quail muscle; however, none of the eight mAbs reacted with either enzyme type from mammalian muscle or from Torpedo electric organ. All eight antibodies showed immunocytochemical localisation of the AChE form at the neuromuscular junctions of chicken twitch muscles.     

Distribution of Epitopes of Trypanosoma cruzi Amastigotes During the Intracellular Life Cycle within Mammalian Cells

ABSTRACT. In this study we have examined the distribution of epitopes defined by monoclonal antibodies raised against Trypanosoma cruzi amastigotes during the intraceullar life cycle of the parasite. We have raised monoclonal antibodies towards amastigote forms and performed preliminary immunochemical characterization of their reactivities. MAB 1D9, 3G8, 2B7, 3B9, and 4B9, and 4B9 react with carbohydrate epitopes of the parasite major surface glycoprotein—Ssp-4 defined by MAB 2C2 [5]: MAB 4B5 reacts with a noncarbohydrate epitope in all developmental stages of the parasite, and MAB 3B2 also detects a noncarbohydrate epitope preferentially in T. cruzi flagellared forms. Vero cells infected with tissue culture-derived trypomastigotes of clone D11 (G strain) were fixed at different times during the intraceullular proliferation of parasites, and processed for immjno-electron microscopy and confocal immunoflurescence with the different monoclonal antibodies. We observed that while the surface distribution of MAB 2C2 and 4B9 epitopes was uniform throughout the cycle, MAB 1D9, 3G8, and 2B7 reacted with cytoplasmic membrance-bound compartments of the amastigotes. MAB 3B9 displayed a unique surface dentate pattern in some amastigotes. MAB 4B5 recognized a curved-shaped structure at the flagellar pocket region in some intracellular amastigotes and localized to the membrane in dividing forms. In intracellular trypomastigotes, MAB 4B5 also displayed a punctate pattern near the flagellar pocket.     

Epitope mapping of monoclonal antibodies for the Deinococcus radiodurans bacteriophytochome

Bacteriophytochromes (BphP) are phytochrome‐like light sensing proteins in bacteria, which use biliverdin as a chromophore. In order to study the biochemical properties of the DrBphP protein, five (2B8, 2C11, 3B2, 3D2, and 3H7) anti‐DrBphP monoclonal antibodies were produced through the immunization of mice with purified full‐length DrBphP and DrBphN (1–321 amino acid) proteins, and epitope mapping was then carried out. Among the five antibodies, 2B8 and 2C11 preferentially recognized the N‐terminal region of BphP whereas 3B2, 3D2, and 3H7 showed preference for the C‐terminal region. We performed further epitope mapping using recombinant truncated BphP proteins to narrow down their target sequences. The results demonstrated that each of the five monoclonal antibodies recognized different regions on the DrBphP protein. Additionally, epitopes of 2B8 and 3H7 antibodies were discovered to be shorter than 10 amino acids (2B8: RDPLPFFPP, 3H7: PGEIEEA). These two antibodies with such specific recognition epitopes could be especially valuable for developing new peptide tags for protein detection and purification.     

The origin of enantioselectivity in aldolase antibodies: crystal structure, site-directed mutagenesis, and computational analysis

Catalytic aldolase antibodies, generated by reactive immunization, catalyze the aldol reaction with the efficiency of natural enzymes, but accept a much broader range of substrates. Two separate groups of aldolase antibodies that catalyze the same aldol reactions with antipodal selectivity were analyzed by comparing their amino acid sequences with their crystal structures, site-directed mutagenesis data, and computational docking of the transition states of the aldol reaction. The crystal structure of aldolase antibody 93F3 Fab' at 2.5A resolution revealed a combining site with two lysine residues, including LysL89 that reacts to form the covalent enamine intermediate. In contrast, antibody 33F12 has one active site lysine, LysH93. The reactive lysine residues in each group of antibodies are differentially located on the heavy and light chain variable regions in pseudo-symmetric opposite orientations, but both within highly hydrophobic environments. Thus, the defining feature for the observed enantioselectivities of these aldolase antibody catalysts is the respective location and relative disposition of the reactive lysine residues within the active sites of these catalysts.     

Epitope mapping of monoclonal antibodies to human plasma membrane Ca2-ATPase

Overlapping fragments of the fourth isoform of human plasma membrane Ca(2+)-ATPase (hPMCA4) and several fragments of hPMCA1 were expressed in bacterial cells and purified by metal affinity chromatography. Enzyme immunoassays of the fragments helped map epitopes for 4 monoclonal antibodies (2D8, 8B8, 7C8 and 5E6). The epitope for 2D8 was localized within the 222-249 site (i.e., in the putative transduction domain), the epitopes for 8B8 and 7C8 were localized within the 330-353 site, in which phospholipids are presumably bound, and the 5E6 epitope was found within the 791-843 site, where the putative hinge region is situated. 2D8 recognizes hPMCA1 and hPMCA4 isoforms, while 8B8 and 7C8 are specific for hPMCA4. The amino acid sequences of these epitopes and phage-displayed mimotopes were compared.     

Modulation of apolipoprotein B antigenic determinants in human low density lipoprotein subclasses

To investigate the effect of low density lipoprotein (LDL) heterogeneity on the conformation of LDL apolipoprotein B (apo-B), the immunoreactivities of 6 monoclonal antibodies against LDL apo-B were measured in 3 LDL subfractions isolated by equilibrium density gradient ultracentrifugation. To ensure a broad range of LDL particles, the LDL subfractions were prepared from normal subjects and patients with hyperapobetalipoproteinemia. With 3 of the antibodies, 1D1, 5E11, and 3A10, LDL fractions 1 (the most buoyant), 2 (the intermediate), and 3 (the densest) were equally immunoreactive and competed similarly with reference whole LDL. In contrast, with 3 other antibodies, 2D8, 3F5, and 4G3, fraction 1 was significantly more reactive than fraction 3; that is for each in turn, 290, 250, and 150% more of the densest LDL protein was required to achieve the same displacement as with fraction 1. Further, the immunoreactivities of the 3 LDL fractions with antibodies 2D8, 3F5, and 4G3 were negatively correlated with their LDL cholesterol to LDL protein ratio with r values of 0.727, 0.898, and 0.870, respectively, suggesting that as LDL particle size decreases, the conformation of the LDL apo-B changes progressively. It is of interest that the antigenic determinants recognized by 3F5 and 4G3 are close to the LDL receptor recognition site on LDL apo-B. Therefore, it is possible that the reduced immunoreactivity of these determinants in dense LDL may be the in vitro correlate of the reduced fractional catabolics rate of dense LDL compared to buoyant LDL previously observed in vivo.     

Monoclonal antibodies against baboon endogenous virus and against host cell antigens.

Monoclonal antibodies were produced by murine hybridomas after immunization with semipurified baboon endogenous virus. In a solid-phase radioimmunoassay, two antibodies (F12-9 and B9-18) reacted with viral antigen only. The antibodies A6-8 and C9-12 also reacted with virus-producing cells but not with control cells, whereas antibodies E4-6 and D12-2 bound to virus-free cells as well. The cytofluorometry technique confirmed these results and showed a competition between antibodies A6-8 and C9-12 for binding to virus-producing cells as well as a competition between antibodies D12-2 and E4-6 for binding to virus-free human cells. An immune precipitation assay with disrupted virions indicated that antibodies A6-8, B9-18, and C9-12 were directed against the gp70 glycoprotein, and that antibody F12-9 reacted with a viral antigen with a molecular weight of 18,000. The syncytia induced in RSa cells by baboon molecular weight of 18,000. The syncytia induced in RSa cells by baboon endogenous virus could be inhibited either when antibody A6-8 or C9-12 was combined to the virus or when the RSa cells were treated with the anticellular antibody D12-2 or E4-6. These two effects were not observed with Mason-Pfizer virus. Thus, of three antibodies with specificities for viral gp70, two (A6-8 and C9-12) were directed at viral sites responsible for syncytium formation. Another antiviral antibody (F12-9) reacted with a protein of unknown function with a molecular weight of 18,000. The two anticellular antibodies were directed at similar or neighboring epitopes, which may be situated within the receptor to the virus.     

Distinct immunohistochemical localization in Kuru plaques using novel anti-prion protein antibodies

By immunizing Prnp-knockout mice with synthetic polypeptides, a panel of mAbs directed to bovine PrP(C) was obtained. The mAb panel was characterized by the ELISA method, where synthetic polypeptides were used for epitope mapping. Different reactivity patterns were identified. The ability of these mAbs to detect abnormal PrP(Sc) in CJD cases was studied by immunohistochemistry. All mAbs were tested for PrP(Sc) in murine, bovine, monkey and human brain tissues. Three mAbs recognized the fragmented PrP epitope in our ELISA. Antibody 1D12 was strongly reactive to ovine and squirrel monkey tissues infected with a scrapie agent, although non-reactive to scrapie-infected mouse tissues. Antibody 2D8 was clearly reactive to type-2 but not type-1 CJD human tissues. Of particular interest was the reactivity of mAb 6C4 with the inner structure of Kuru plaques (peripheral pattern) in a type-2 CJD case and mAb T2, 1D12, 2B11, 2D8, 4B5 and 6G3-2 with the central area (central pattern). The fact that different anti-PrP mAbs possess distinct staining properties suggests that the PrP(c) to PrP(Sc) conversion might involve a multiple-step process.     

Production of Monoclonal Antibodies to Guinea Pig Liver Transglutaminase

Monoclonal antibodies are now a powerful tool in biology and medicine. Transglutaminase has been implicated in diverse biological functions, and the characteristics of its catalytic action are suitable for applied enzymology. In this study, we produced hybridoma cells which synthesize monoclonal antibodies against guinea pig liver transglutaminase by fusing mouse myeloma cells with spleen cells of mouse immunized with the enzyme protein. Eight hybridoma clones (coded 2F, 4B, 7C, 8B, 8D, 8E, 9F and 11C) were selected to produce monoclonal antibodies. The subclass of IgG produced by clone 9F was IgG_2a and those from the seven other clones were all IgG₁ The 9F antibody inhibited transglutaminase activity, but the other antibodies did not. A solid-phase antibody-binding assay showed that of these antibodies, 8D antibody has the highest affinity to the antigen. Transglutaminase protein in crude liver extract was identified with Western blotting analysis using 8D antibody as the probe.     

Use of monoclonal antibodies to human lymphocytes to identify lymphocyte subsets in lymph nodes of the rhesus monkey and the dog

Using commercially available monoclonal antibodies that bind to human lymphocyte subsets, we examined lymph nodes from the rhesus monkey and the dog for their binding ability to these monoclonal antibodies. The avidin biotin-peroxidase immunostaining procedure was used, and the following antibodies were reactive in the rhesus monkey: Leu 4, Leu 3a, OKT4, Leu 2a, OKT8, T8, Leu 5b, T11, Leu 14, B1, and Leu 12. No immunoreactivity was observed in the dog lymph node except for moderate reactivity of OKT8. The following antibodies failed to react in both the rhesus monkey and the dog: OKT3, T1, T2, T1B, T4, T8A, Pan B, and T29/33. Kappa and lambda immunoglobulin light chains were positive in both the dog and monkey.