Purification and Characterization of Canine Serum Ferritin-binding Proteins

Ferritin-binding protein (FBP) is known to interact with circulating ferritins in mammals. Canine FBPs were purified from canine serum by affinity chromatography and were identified as IgM, IgG, and IgA by immunoblotting with alkaline phosphatase-labeled antibodies to canine IgM, IgG, and IgA heavy chains. Following further purification by application to a Sephacryl S-300 column, canine FBPs were separated into 81.3- and 27.7-kDa bands by sodium dodecyl sulfate-polyacryamide gel electrophoresis, and the 81.3-kDa band reacted with the anti-canine IgM heavy chain antibody. Purified canine FBP bound to canine liver ferritin, but not to canine albumin and transferrin. FBP showed greater binding to the expressed bovine ferritin H-chain homopolymer than to the expressed bovine ferritin L-chain homopolymer. The binding of FBP with canine liver ferritin was dose-dependently inhibited by anti-rat liver ferritin antibody, and the anti-ferritin antibody dissociated the bound FBP in a dose-dependent manner, even after binding FBP with liver ferritin. The canine ferritin H subunit peptide fragment with amino acid residues 148–155 (NH₂-GDHVTNLR-COOH) in its C-terminal region was recognized by FBP. These results indicate that canine serum FBPs are autoantibodies to ferritin (IgM, IgG, and IgA) and that anti-ferritin autoantibody (IgM) recognizes the C-terminal region of ferritin H subunit.     

Characterization of feline serum ferritin-binding proteins: the presence of a novel ferritin-binding protein as an inhibitory factor in feline ferritin immunoassay

Ferritin-binding proteins (FBPs) such as anti-ferritin antibody, α-2-macroglobulin, apolipoprotein B are expected to interact with circulating ferritin to eliminate it from circulation. However, we found that feline serum more strongly inhibits the detection of canine liver ferritin by immunoassay than its apoferritin; putative FBPs probably conceal ferritin epitopes detected by anti-ferritin antibodies. After complex formation between affinity-purified FBPs and canine liver ferritin, co-immunoprecipitates of the complex by anti-bovine spleen ferritin antibody were found to contain autoantibodies (IgG, IgM, and IgA) to ferritin by immunoblot analysis with antibodies specific for feline IgG, IgM, and IgA. On the other hand, affinity-purified samples did not show any inhibitory effect in the ferritin immunoassay. This result shows that feline serum has another FBP, which inhibits ferritin immunoassays, but not anti-ferritin autoantibody. A feline FBP was partially purified from feline serum by (NH₄)₂SO₄ fractionation (33–50%), gel filtration chromatography, and anion exchange chromatography. After binding of the partially purified sample with canine liver ferritin coupled-Sepharose gel, the FBP was separated and purified from complexes formed in a native-PAGE gel. SDS–PAGE analysis showed that the purified FBP is a homomultimer composed of 31 kDa monomeric subunits connected by intermolecular disulfide bonds. Detection of feline liver ferritin by immunoassay was inhibited by FBP in a dose-dependent manner. The purified protein molecules appeared to be conglomerate of pentraxin-like molecules by its electron micrographic appearance. These results demonstrate that feline serum contains a novel FBP as inhibitory factor of ferritin immunoassay with different molecular properties from those of other mammalian FBPs, in addition to auto-antibodies (IgG, IgM, and IgA) to ferritin.     

Characterization of ferritin and ferritin-binding proteins in canine serum

Ferritin and ferritin-binding proteins in canine serum were characterized. A certain percentage of ferritin in canine serum, but no tissue ferritin, was precipitated by centrifugation at 16,000×g for 30 min. The precipitated ferritin was found to contain two subunits corresponding to the H and L subunits of canine liver ferritin by immunoblotting, the H subunit being predominant. More ferritin was precipitated from canine sera which had been incubated with anti-rat liver ferritin antibody than from untreated sera, and the H chain also predominated. To evaluate the possibility that the autoantibody was responsible for the precipitation of canine serum ferritin, the ferritin-binding activities of canine antibodies were examined using liver ferritin-coated microtiter plates and alkaline phosphatase-labeled antibodies specific for canine IgM, IgA, and IgG heavy chains. The results showed that IgM and IgA, but not IgG, had considerable ferritin-binding activities. Given these results, we suggest that there is H-chain-rich isoferritin in canine serum, and that ferritin exists as an immune complex.     

The isolation and properties of multiple forms of folate binding protein in cultured KB cells

The folate binding proteins (FBPs) of KB cells which were cultured in normal (N) and folate-deficient (D) medium have been characterized. The 200,000 g supernate of lysed cells contained two FBPs which could be separated by DEAE-Bio-Gel A chromatography, indicating that they differ in ionic charge although they could not be separated by gel filtration through Sephadex G-100 (apparent Mr approximately 40,000). Two species of FBP, a major form of apparent Mr approximately 160,000 and a minor form of apparent Mr approximately 40,000, were identified by gel filtration through Sephadex G-150 in the membrane component of the cells after solubilization with Triton X-100. An additional FBP was isolated and purified by affinity chromatography from the medium in which these cells were cultured. By gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent Mr of this FBP was approximately 44,000. The association constants for pteroylglutamic acid of the FBPs in the 200,000 g cell lysate supernate, culture medium, and Triton-solubilized membrane were similar and the relative affinity of folate analogs for the FBP, vis-à-vis pteroylglutamic acid, was similar for all species. An antiserum raised to the purified FBP from the culture medium precipitated the FBPs in the 200,000 g cell lysate supernate, Triton-solubilized membrane, and culture medium, indicating antigenic homology among these FBPs. There was no unsaturated FBP in the 200,000 g cell lysate supernate or medium when KB cells were cultured in N medium. However, when cells were cultured in D medium, the unsaturated FBP of the 200,000 g cell supernate and culture medium was substantial (9.2 and 14.1 pmol/mg protein, respectively). Unsaturated FBP was detected in the membrane of normal cells but this also increased when these cells were cultured in D medium (4.5 to 756 pmol/mg protein), indicating that the FBPs of these cellular compartments are normally saturated by folate. After 16 weeks of culture in D medium, the total folate binding capacity of the membrane-associated FBP was twofold greater than that of normal KB cells, indicating the induction of FBP.     

Physiological implications of mammalian ferritin-binding proteins interacting with circulating ferritin and a new aspect of ferritin- and zinc-binding proteins

Serum ferritin levels are relatively low (<1 µg/ml) and serum ferritin generally disappears rapidly from the circulation (t _1/2 < 10 min). There are various mammalian ferritin-binding proteins (FBPs) in the blood. Ferritin is cleared by direct uptake by ferritin receptors and by indirect receptor-mediated uptake of FBP complexed with ferritin. Mammalian ferritin binds both heme and iron, and binding occurs through two mechanisms: direct binding with ferritin to H-kininogen and anti-ferritin autoantibody, and indirect heme-mediated binding of fibrinogen and apolipoprotein B to ferritin. Anti-ferritin autoantibody and fibrinogen are proposed to be common mammalian FBPs, as is α₂-macroglobulin. FBP-ferritin binding may affect blood coagulation and influence iron metabolism, oxidative condition, angiogenesis, inflammatory condition and immune response. Aside from apolipoprotein B, FBPs bind zinc ion to form antioxidant and anti-inflammatory agents. The possible simultaneous uptake of zinc ion with FBP-ferritin complex is likely to attenuate iron- and/or heme-mediated oxidative damage and inflammatory response.     

Megalin binds and mediates cellular internalization of folate binding protein

Folate is an essential vitamin involved in a number of biological processes. High affinity folate binding proteins (FBPs) exist both as glycosylphosphatidylinositol-linked, membrane associated folate binding proteins and as soluble FBPs in plasma and some secretory fluids such as milk, saliva and semen. The function and significance of FBPs are unresolved, however, it has been suggested that they may facilitate folate uptake, e.g. during suckling. The present study shows that megalin, a large, multiligand endocytic receptor and member of the low-density lipoprotein-receptor family, is able to bind and mediate cellular uptake of FBP. Surface plasmon resonance analysis shows binding of bovine and human milk FBP to immobilized megalin, but not to low density lipoprotein receptor related protein. Binding of (125)I-labeled folate binding protein (FBP) to sections of kidney proximal tubule, known to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is inhibited by receptor associated protein and by antimegalin antibodies. Microinjection of (125)I-labeled FBP into renal tubules in vivo shows proximal tubular uptake by endocytosis. Megalin is expressed in several absorptive epithelia, including intestine and kidney proximal tubule, and thus the present findings provide a mechanism for intestinal and renal endocytic uptake of soluble FBP.     

Detection of Foot-and-Mouth Disease Virus Antibodies: II. Use of Fractionated Bovine Antisera for Improving the Specificity of a “Passive” Hemagglutination Test1

Because 7S immunoglobulin (Ig) G antibodies of low type specificity were present in mixtures with highly specific 19S IgM antibodies, many bovine antisera to foot-and-mouth disease virus (FMDV) type A(12), strain 119 cross-reacted with type O of FMDV and to some degree with type C in the passive hemagglutination (HA) test. After 19S IgM antibodies were separated by density gradient centrifugation or precipitated with 4% (w/v) polyethylene glycol, the antigen could be determined with "block" HA tests. Such tests used several antigen concentrations in the titration of each antiserum. Adding 4% (w/v) polyethylene glycol to the serum was especially convenient for rapid precipitation of 19S IgM antibodies for the test. Similar results were obtained with bovine 19S IgM antibodies to FMDV type O, subtype 1, strain Caseros and type C strain Rezende.     

Monoclonal antibodies against bovine growth hormone.

A number of mouse x mouse hybridomas producing monoclonal antibodies (MAbs) against bovine growth hormone (bGH) were prepared by fusion of spleen cells from bGH-primed mice (Balb/c) with non-secretory mouse myeloma cells (PAIOP3) and characterized. MAbs obtained from three fusion experiments belonged to IgM, IgG1 and IgG2b class/subclass of antibodies. Cross-reaction studies indicated that generated antibodies were against three different epitopes of bGH. VIA6E8 (IgG1) and VIIB2E11C9 (IgM) did not cross-react with ovine prolactin (oPRL), ovine leutinizing hormone (oLH) and porcine follicle stimulating hormone. Antibody VIB3C9E8 (IgM) exhibited cross-reaction with oPRL and oLH. Antibody VIC1F9 (IgG2b) cross reacted with oPRL. All MAbs were against conformational epitopes of bGH.     

Characterization of autoantibodies to ferritin in canine serum

Ferritin-binding proteins circulating in mammalian blood are thought to be involved in the clearance of ferritin. The present study characterizes canine serum autoantibodies (IgM and IgA) that react with ferritin. Canine IgM and IgA bound to bovine spleen ferritin as well as canine liver ferritin. To examine the specificity of canine IgM and IgA to ferritin H and L subunits, we used canine heart ferritin and canine liver ferritin with H/L subunit ratios of 3.69 and 0.43, respectively. Canine IgM and IgA recognized both of the H- and L-subunit-rich isoferritins, showing that their binding activities to ferritin depend on the H-subunit content. Recombinant bovine H-chain ferritin homopolymer expressed in a baculovirus expression system bound more with IgM and IgA than the recombinant L-chain homopolymer expressed under the same conditions. These results suggest that canine IgM and IgA recognize H-subunit-rich isoferritins, and that H-subunit-rich isoferritins are cleared from the circulation more rapidly than L-subunit-rich isoferritins.     

IgG autoantibody activity in normal mouse serum is controlled by IgM

In the serum of normal BALB/c mice, IgG antibody reactivity to mouse actin and tubulin, DNA, and TNP groups was very low compared to that of the IgM. This activity was considerably increased when IgG was separated, by affinity chromatography on protein A-Sepharose, whereas no difference in the IgM activity was observed. Addition of IgM to IgG isolated from the same serum resulted in the inhibition of IgG binding to these Ag. Isolation of IgG antibodies on actin, TNP, and tubulin immunoadsorbents has indicated that at least part of the IgG antibodies is polyreactive. In order to understand this inhibition better, experiments with F(ab')2 fragments of IgG were performed. IgM inhibited the binding of F(ab')2 to the antigens in a dose-dependent manner and reacted with immobilized F(ab')2. IgM isolated on F(ab')2 immunoadsorbent, as compared to the initial IgM preparation, were less active toward the Ag but more inhibitory for IgG binding to the Ag. In some pathologic situations, IgM failed to inhibit some IgG antibody activities. The anti-DNA IgG activity from (NZB x NZW)F1 mice was not affected by autologous IgM. Similarly the anti-tubulin IgG from mice infected with Trypanosoma cruzi were less inhibited by IgM from autologous serum than antitubulin IgG from normal mice. These results are compatible with the existence in normal mice of an idiotypic-like network, regulating via an IgM population in the serum, the binding of IgG autoantibodies to self Ag. Modifications of this idiotype-anti-idiotype system might lead to the expression and/or expansion of autoreactive IgG-producing clones.     

Analysis of folate binding protein N-linked glycans by mass spectrometry

The folate binding protein (FBP), also known as the folate receptor (FR), is a glycoprotein which binds the vitamin folic acid and its analogues. FBP contains multiple N-glycosilation sites, is selectively expressed in tissues and body fluids, and mediates targeted therapies in cancer and inflammatory diseases. Much remains to be understood about the structure, composition, and the tissue specificities of N-glycans bound to FBP. Here, we performed structural characterization of N-linked glycans originating from bovine and human milk FBPs. The N-linked glycans were enzymatically released from FBPs, purified, and permethylated. Native and permethylated glycans were further analyzed by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS), while tandem MS (MS/MS) was used for their structural characterization. The assignment of putative glycan structures from MS and MS/MS data was achieved using Functional Glycomics glycan database and SimGlycan software, respectively. It was found that FBP from human milk contains putative structures that have composition consistent with high-mannose (Hex(5-6)HexNAc(2)) as well as hybrid and complex N-linked glycans (NeuAc(0-1)Fuc(0-3)Hex(3-6)HexNAc(3-5)). The FBP from bovine milk contains putative structures corresponding to high-mannose (Hex(4-9)HexNAc(2)) as well as hybrid and complex N-linked glycans (Hex(3-6)HexNAc(3-6)), but these glycans mostly do not contain fucose and sialic acid. Glycomic characterization of FBP provides valuable insight into the structure of this pharmacologically important glycoprotein and may have utility in tissue-selective drug targeting and as a biomarker.     

A calcium-dependent cryoglobulin IgM kappa/polyclonal IgG.

The mechanisms responsible for cold-induced precipitation of mixed cryoglobulins are not well understood. A mixed cryoglobulin IgM kappa/IgG (type II) of a patient with Sj?gren's syndrome was studied because of its unique properties. This cryoglobulin precipitated in serum but not in serum containing 10 mM EDTA. The cryoprecipitation was shown to require calcium (Ca) and was optimal at 1 mM of free Ca. Cryoprecipitation was also induced by Ba, Mn, and Sr, but not by Mg and Co. Purified IgM kappa/IgG complexes precipitated in the presence of Ca, but not IgM kappa alone. There was no significant binding of 45Ca to the purified IgM kappa, IgM kappa/IgG complexes formed with purified components, and the cryoprecipitate. The relative affinity of the radiolabeled [125I]IgM kappa for IgG was 3.6 x 10(3) liters/mol at 37 degrees C as assessed by sucrose density gradient ultracentrifugation, and increased to 1.7 x 10(4) liters/mol at 4 degrees C. The addition of Ca produced no change in the affinity at 37 degrees C and 4 degrees C. The absence of a direct effect of Ca on the Ag/antibody reaction was confirmed in experiments using polyethylene glycol as precipitating agent. In conclusion, two independent steps were responsible for the precipitation of this cryoglobulin. The first step was an efficient formation of soluble immune complexes as produced by a drop in temperature. The second step was caused by a change in the physicochemical conditions--the presence of Ca--which induced polymerization of the IgM kappa/IgG complexes.     

Identification of a Mg2+-dependent protease in human placenta which cleaves hydrophobic folate-binding proteins to hydrophilic forms

Hydrophobic folate-binding proteins (FBPs), which are only 5-10 kDa larger than 40-kDa hydrophilic FBPs, bind significant quantities of Triton X-100 micelles and elute as apparent 160-kDa species on Sephacryl S-200 gel filtration in Triton X-100. Detergent-solubilized placental membranes release a major (greater than 95%) 40-kDa hydrophilic FBP species as well as a minor apparent 160-kDa folate binding species when similarly analyzed. We tested the hypothesis that this recovery of predominantly hydrophilic FBPs was mediated by a putative hydrophobic FBP-specific placental protease. When placenta was solubilized in the presence of increasing concentrations of EDTA, there was a progressive increase in apparent 160-kDa folate binding moieties concomitant with a decrease in 40-kDa FBPs. At 20 mM EDTA, a single apparent 160-kDa folate binding species was recovered and the 40-kDa FBPs could not be detected by radioligand binding or specific radioimmunoassay. The apparent 160-kDa species specifically bound radiolabeled folates and were specifically immunoprecipitated by rabbit anti-40-kDa FBP antiserum. On 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose and probing with anti-40-kDa FBP antiserum, the apparent 160-kDa FBPs electrophoresed as 45-kDa species. Detergent binding studies indicated that apparent 160-kDa FBPs were hydrophobic, thus accounting for the molecular weight discrepancy in gel filtration in Triton X-100 versus sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The EDTA-mediated inhibition of conversion of hydrophobic FBPs to hydrophilic FBPs by protease was reversed in a dose-dependent manner by Mg2+. If this protease is physiologically relevant, it could play an important regulatory role in folate transport by influencing the net number of hydrophobic FBPs on the cell surface.     

Normal human sera contain antibodies directed at Fab of IgA

Serum samples from 26 normal volunteers were evaluated by isotype-specific ELISA for the presence of IgG and IgM antibodies directed at IgA. Although there were wide variations in antibody levels, anti-IgA antibodies of both isotypes were found in all individuals tested. The anti-IgA activity was detected against a variety of polymeric and monomeric IgA1 and IgA2 myeloma proteins containing both kappa and lambda light chains. By using Fab and Fc fragments generated by incubation of an IgA1 myeloma protein with IgA1 protease, it was shown that the anti-IgA activity was specific for the Fab portion of the IgA molecule. It was also demonstrated that the serum of two individuals contained both IgG and IgM activity directed at autologous affinity-purified IgA. IgM antibody levels against both whole IgA and Fab of IgA were significantly higher than IgG antibody levels. Cells producing anti-IgA antibodies of both isotypes were detected in lipopolysaccharide-stimulated human spleen.     

Identification of FUSE-binding proteins as interacting partners of TIA proteins

TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm.     

The localization of the antibody response in milk or bile depends on the nature of the antigen

Immunization in the Peyer's patches of rats with horse spleen ferritin or Escherichia coli 06 carrying type 1 pili resulted in an IgA antibody response detected in milk and bile and an IgG and IgM antibody response in serum, milk, and bile. The IgA antibody response to type 1 pili was as a mean 5.0-fold higher in milk than in bile. In contrast IgA antibody activity to 06 LPS was as a mean 6.3-fold higher in bile than in milk. The IgA antibodies to ferritin were randomly distributed between milk and bile. The IgG and IgM antibody activity to all three antigens studied were higher in the milk than in the bile. The secretory antibody response could be transferred from immunized rats to unimmunized rats with mesenteric lymph node cells (MLN) taken from donor rats 4 days after immunization in the Peyer's patches. IgA antibodies to pili and ferritin appeared solely in the milk of the recipients, whereas IgA antibodies to the 06 LPS only appeared in the bile. The ratios serum:milk and serum:bile for the IgG and IgM antibodies indicated an antigen-specific direction of homing with local production of these two isotypes primarily in the mammary gland. Antibody-forming cells of the IgA class could not be detected in the MLN on the day the cells were transferred. It is concluded that the difference seen in antibody distribution between milk and bile is not due to dissemination of antigen, but instead a result of different homing or expansion at the mucosal-glandular site dependent on the antigen specificity of the migrating cells.     

The prevalence of Toxoplasma gondii antibodies in wild and captive cetaceans from Japan

The seroprevalence of Toxoplasma gondii was investigated in wild and captive cetaceans from Japan. Antibodies against T. gondii were examined by both latex agglutination test (LAT) and indirect hemagglutination test (IHAT) for 77 serum or plasma samples obtained from 59 individuals of 6 species, including 2 hybrids. Antibody titers greater than 1:64 in LAT and greater than 1:640 in IHAT, indicative of the presence of T. gondii, were found in 11.9% of 59 individuals. In 7 samples that showed a positive reaction by IHAT, T. gondii titers were examined for each immunoglobulin (Ig) fraction separated by sucrose gradient centrifugation. The antibody peaks in each fraction were divided into 3 types, thought to be a reaction of IgM (type 1), IgG (type 2), and IgM with IgG (type 3). Type 1 was found in serum from a bottle-nosed dolphin (Tursiops truncatus) and a killer whale (Orcinus orca) sampled soon after capture off the Japanese coast in 1988; it was concluded that infection in the wild had occurred less than 15 yr before the study was performed. The prevalence of putative IgM and IgG antibodies from a captive-bred T. truncatus suggested that T. gondii infection also occurred in the aquarium.     

Production of auto-anti-idiotypic antibody during the normal immune response. XII. Enhanced auto-anti-idiotypic antibody production as a mechanism for apparent B-cell tolerance in rabbits after feeding antigen

Rabbits fed trinitrophenylated bovine serum albumin (TNP-BSA) generated fewer anti-TNP plaque-forming cells but greater numbers of hapten (TNP)-augmentable IgM and IgG PFC following immunization with TNP-Ficoll or TNP-Brucella abortus than did animals not previously fed antigen. Spleen and mesenteric and bronchial lymph nodes were similarly affected. In addition more auto-anti-idiotype (Id) antibody (anti-anti-TNP) was eluted by hapten from spleen cells of antigen-fed rabbits than from spleen cells of control rabbits not prefed antigen. Gel filtration studies ruled out the possibility that the Id binding activity in the eluates was due to immune complexes. The isotype of the anti-Id was IgG except in one rabbit where it was IgM. The results are consistent with the interpretation that the production of auto-anti-Id antibody is one of the factors responsible for the specific depression of the IgM and IgG immune responses which follows antigen feeding. In contrast the antigen feeding resulted in priming for an IgA anti-TNP response without detectable hapten-augmentable IgA PFC.     

Development and characterization of monoclonal antibodies to foot and mouth disease virus type 'C'

Five fusion experiments were conducted with spleen cells from Balb/c mice immunized with purified 146S antigen of foot and mouth disease virus type 'C' (vaccine strain). Monoclones (31) thus developed were isotyped as IgM (3), IgG1 (6), IgG2a (5), IgG2b (3) and IgG3 (14). Eleven clones isotyped as IgM, IgG2a and IgG2b showed neutralizing activity in virus neutralization and plaque reduction tests. Six of the neutralizing clones precipitated 146S virus in Ouchterlony reaction. On the basis of location of MAb reactive epitopes in relation to intact virus (146S), 12S particles and VP1 in ELISA test, the clones were classified as Class II (6), Class III (11) and Class IV (14). These clones may be useful for purposes of antigen detection from field isolates and for estimation of antibody titres in vaccinated animals.     

Production and characterization of a bovine T cell-specific monoclonal antibody identifying a mature differentiation antigen

A monoclonal antibody (MAb), BLT-1, with specificity for bovine mature T cells was prepared by somatic cell hybridization of myeloma NS-1 and spleen cells from BALB/c mice hyperimmunized with bovine T lymphocytes. The MAb reacted with over 92% of nylon wool-nonadherent lymphocytes (T cells) but not with nylon wool-adherent EAC-positive lymphocytes (B cells) in the indirect immunofluorescence assay. It is an IgM, with kappa-light chains, which fixed complement well and killed over 95% of mature T cells in complement-mediated cytotoxicity assays. It reacted with the same proportions of peripheral lymphoid cells (peripheral blood, lymph nodes, and spleen) as the polyclonal goat anti-bovine thymocyte serum (GABTS), but only with 25% of GABTS-positive thymocytes. Immunoperoxidase staining of frozen tissue sections showed that the BLT-1-positive cells were located in the medulla of the thymus and in the T lymphocyte areas of lymph nodes. Western immunoblotting assays showed that the BLT-1-reactive membrane antigen is a 22,000 m.w. protein which was inducible in bovine thymocytes with bovine thymic hormones, thymosin fraction 5, thymosin alpha 1, and thymopentin ORF-18150, indicating that it is a mature T lymphocyte differentiation antigen. The thymosin alpha 1 and thymopentin were found to show additive effects on mature T cell antigen expression by bovine thymocytes.