Production,cross reactivity,and epitope analysis of monoclonal antibodies against rat kidney gamma glutamyltransferase

Eighteen IgGl monoclonal antibodies (blabs) have been produced against gamma-glutamyl transferase (GGT) from rat kidney. They were specific to the light subunit of the enzyme with affinity constants ranging from 0.3 to 7.5 10⁸ M^–1, while they did not react with GGT from other sources i.e. human and pig kidney, rat and guinea pig liver, suggesting species and organ specificity. Two of the blabs (N° 11 and 21) lost their immunoreactivities towards rat kidney GGT in the presence of N-acetyl-neuraminic acid, while immunoreactivities of the other blabs were unchanged. Furthermore, Mabs No 11 and 21 did not react with desialylated rat kidney GGT. These findings suggest that N-acetyl-neuraminic acid is involved in the epitopes recognized by these two Mabs.Abbreviations ELISA enzyme linked immunosorbent assay - GGT gamma-glutamyltransferase - Mab monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Stromelysin, a connective tissue-degrading metalloendopeptidase secreted by stimulated rabbit synovial fibroblasts in parallel with collagenase. Biosynthesis, isolation, characterization, and substrates

Rabbit synovial fibroblasts induced to undergo a specific switch in gene expression by agents that alter cell morphology secreted the neutral proteinase precursor procollagenase (apparent Mr of 53,000 and 57,000). A major Mr = 51,000 polypeptide that was always induced coordinately with procollagenase has now been identified as the proenzyme form of a metal-dependent proteinase active at neutral pH. We have named this proteinase stromelysin. Prostromelysin and procollagenase were the most prominent [35S]methionine-labeled secreted proteins of the induced fibroblasts. By the use of casein degradation as an assay for enzyme activity, stromelysin was isolated with high yield from the conditioned culture medium of 12-O-tetradecanoylphorbol 13-acetate-treated fibroblasts and migrated as an active form of Mr = 21,000 that was immunologically identical to the proteoglycan-degrading proteinase purified from rabbit bone. Immunoglobulin G from antiserum raised to purified rabbit bone proteoglycanase immunoprecipitated the Mr = 51,000 proenzyme form from conditioned medium of induced rabbit cells and also immunoprecipitated an Mr = 55,000 polypeptide from induced human fibroblasts. When rabbit prostromelysin was activated by trypsin or 4-aminophenylmercuric acetate, the proenzyme was converted to an active form of Mr = 41,000. During the course of the purification, prostromelysin was converted to an additional activatable form of Mr = 35,000 and additional active forms of Mr = 21,000-25,000, which had related peptide maps distinct from collagenase. All of these forms were immunologically cross-reactive. Purified stromelysin degraded casein, cartilage proteoglycans, fibronectin, alpha 1-proteinase inhibitor, and immunoglobulin G2a and had limited activity on laminin, elastin, type IV collagen, and gelatin, but did not degrade type I collagen. Stromelysin was inhibited by EDTA, 1,10-phenanthroline, and the specific glycoprotein tissue inhibitor of metalloproteinases isolated from human amniotic fluid and was therefore classified as a metalloproteinase.     

Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin)

The mechanism of activation of tissue procollagenase by matrix metalloproteinase 3 (MMP-3)/stromelysin was investigated by kinetic and sequence analyses. MMP-3 slowly activated procollagenase by cleavage of the Gln80-Phe81 bond to generate a fully active collagenase of Mr = 41,000. The specific collagenolytic activity of this species was 27,000 units/mg (1 unit = 1 microgram of collagen digested in 1 min at 37 degrees C). Treatment of procollagenase with plasmin or plasma kallikrein gave intermediates of Mr = 46,000. These intermediates underwent rapid autolytic activation, via cleaving the Thr64-Leu65 bond, to give a collagenase species of Mr = 43,000 that exhibited only about 15% of the maximal specific activity. Similarly, (4-aminophenyl)mercuric acetate (APMA) activated procollagenase by intramolecular cleavage of the Val67-Met68 bond to generate a collagenase species of Mr = 43,000, but with only about 25% of the maximal specific activity. Subsequent incubation of the 43,000-Mr species with MMP-3 resulted in rapid, full activation and generated the 41,000-Mr collagenase by cleaving the Gln80-Phe81 bond. In the case of the proteinase-generated 43,000-Mr species, the action of MMP-3 was approximately 24,000 times faster than that on the native procollagenase. This indicates that the removal of a portion of the propeptide of procollagenase induces conformational changes around the Gln80-Phe81 bond, rendering it readily susceptible to MMP-3 activation. Prolonged treatment of procollagenase with APMA in the absence of MMP-3 also generated a 41,000-Mr collagenase, but this species had only 40% of the full activity and contained Val82 and Leu83 as NH2 termini. Thus, cleavage of the Gln80-Phe81 bond by MMP-3 is crucial for the expression of full collagenase activity. These results suggest that the activation of procollagenase by MMP-3 is regulated by two pathways: one with direct, slow activation by MMP-3 and the other with rapid activation in conjunction with tissue and/or plasma proteinases. The latter event may explain an accelerated degradation of collagens under certain physiological and pathological conditions.     

Tissue distribution of two major components of synaptonemal complexes of the rat

In this paper we describe an analysis of the tissue distribution of two recently identified components of synaptonemal complexes (SCs), an Mr 125000 and an Mr 190000 protein, in the male rat by immunoblot analysis and immunocytochemical techniques. We compared the tissue distribution of these antigens with that of two earlier identified SC components, an Mr 30000 and an Mr 33000 polypeptide. For this purpose we used monoclonal antibodies (Mabs) that react exlusively with SCs in lysed spermatocytes, and that recognize the above mentioned antigens specifically in immunoblots of SC proteins or of nuclear proteins from spermatocytes: these were Mab IX9D5 (anti-190000), Mab IX5B2 (anti-125000), Mab II52F10 (anti-30000+33000), and Mab IX8G9 (anti-30000+33000). In the immunoblot experiments, we could detect the Mr 190000 and 125000 antigens exclusively in blots of SC proteins or nuclear proteins from spermatocytes; these antigens were not detectable in blots of nuclear proteins from liver, brain, spermatogonia or spermatids or in blots of proteins from mitotic chromosomes or nuclear laminae. With the anti-30000+33000 Mabs we obtained essentially the same result, except that Mab IX8G9, but not II52F10, recognizes a small amount of Mr 30000 antigen in blots of nuclear proteins from spermatids and spermatogonia. Although this might be ascribed to contamination of the isolated spermatids and spermatogonia, we cannot exclude that a small amount of Mr 30000 antigen is present in these cells. In the immunofluorescence analysis, the testis was the only tissue that reacted detectably with the above antibodies. Within the testis, spermatocytes and some early spermatids were the only cell types that contained detectable amounts of antigen. The Mr 125000 antigen was exclusively observed in nuclei of spermatocytes, from zygotene up to and including diplotene, in paired segments of SCs. The Mr 30000+33000 and 190000 antigens were present in paired as well as unpaired segments of SCs in nuclei of permatocytes, from zygotene up to and including diplotene and in the nuclei of some early spermatids in presumed remnants of SCs. We conclude that SCs consist largely of meiosisspecific proteins.by U. Scheer     

Monoclonal antibodies against different epitopes of a 40 Kd capsid protein of infectious bursal disease viruses.

Nine monoclonal antibodies (Mab) against a 40 Kd capsid protein of infectious bursal disease virus (IBDV) strain P3009 were isolated. They were characterized by enzyme-linked immunosorbent assay, indirect fluorescent antibody staining and virus neutralization. They were divided into two groups concerning virus neutralization. Group I Mabs were able to neutralize virus infectivity; however, group II Mabs were not. Competitive binding assays using these Mabs demonstrated the existence of two distinct antigenic regions (A and B) on the 40 Kd protein. Region A was recognized by group I Mabs and region B was by group II Mabs. The binding reaction with group I Mabs was affected by denaturing of the viral proteins, indicating that the antigenic region involving neutralization was conformation-dependent. The results of enzyme-linked immunosorbent assays and virus neutralization tests suggested that group I Mabs might react with one epitope within region A and group II Mabs with 2 or 3 epitopes within region B.     

Collagenase is a major gene product of induced rabbit synovial fibroblasts

We have investigated the effects of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), on rabbit synovial fibroblasts, and found that this agent induced a major switch in gene expression in these cells that was marked by the specific induction of the neutral proteinase, collagenase, and was always accompanied by alterations in cell morphology. Procollagenase synthesis and secretion was first observed 6-12 h after the addition of TPA. The rate of collagenase production (1-5 U, or approximately 0.2-1 micrograms secreted procollagenase protein per 10(5) cells per 24 h) depended on the TPA concentration (1-400 ng/ml) and time of exposure (1-72 h). Procollagenase was the most prominent protein visible by direct silver staining or by autoradiography after SDS PAGE of [35S]methionine-labeled proteins. The two procollagenase bands of Mr 53,000 and 57,000, which migrated as a family of spots on two-dimensional gels and were immunoprecipitated by antibodies to purified rabbit collagenase, accounted for 23% of the newly synthesized, secreted protein in TPA-treated cells. Cell-free translation of mRNA from TPA-treated cells in rabbit reticulocyte lysate produced a single band of immunoprecipitable preprocollagenase (Mr 55,000) as a major product (5% of total) that migrated as a single spot on two-dimensional gels. Secreted procollagenase, preprocollagenase , and active collagenase (purified to homogeneity; specific activity 1.2 X 10(4) U/mg protein) had related peptide maps. Two other major secreted proteins, a neutral metalloproteinase of Mr 51,000 and a polypeptide of Mr 47,000, were also induced by TPA. In contrast to the induction of these four polypeptides, TPA decreased synthesis and secretion of a number of proteins, including collagen and fibronectin. Thus, collagenase is a convenient marker for major alterations in the pattern of protein synthesis and secretion by rabbit synovial fibroblasts treated with TPA.     

Collagenase, procollagenase and bone resorption. Effects of heparin, parathyroid hormone and calcitonin.

1. The addition of heparin to the culture fluid of mouse tibiae or calvaria did not cause any significant resorption of bone collagen or mineral. However, heparin (or analogue sulfated polyanions), enhanced greatly the amount of latent, trypsin-activatable collagenase (i.e. procollagenase) released by the bones in the medium without influencing that of directly active collagenase which was always very low. Heparin appeared to act by increasing the production of the enzyme which is immediately excreted. Procollagenase and collagenase are not stored in bone tissue, even under conditions where it is in active resorption. 2. Parathyroid hormone induced in the explants a resorption of both mineral and collagen that was inhibited by calcitonin. These hormones, however, had no influence on the release of procollagenase or collagenase either in the presence or in the absence of heparin. 3. Once activated, bone collagenase digested the collagen of the bone explants, and more extensively after their demineralization. Thus the latent collagenase that accumulates around non-resorbing bones has to be considered as a precursor, (and not as a residue), of active enzyme. 4. Active collagenase added to incipient cultures of bones disappeared with a half-life of 24 h. The lost enzyme could, however, not be reactivated by trypsin and thus was not transformed into latent procollagenase.     

Properties of a panel of monoclonal antibodies which react with the human T cell antigen receptor on the leukemic line HPB-ALL and a subset of normal peripheral blood T lymphocytes

Five Mab raised against the T cell antigen receptor of the human T cell line HPB-ALL which react with a subpopulation of normal peripheral blood T cells are described. Three Mab, 3D6, 1C1, and 1C2, react with 3 to 5% of normal PBL and stimulate proliferation of the cells with which they react. An increase in the number of cells which react with all five Mab occurs. Two Mab, 2D4 and 65, react with subsets of the cells which bind 1C1, 1C2, and 3D6 and divide the family into four subgroups, 2D4+ 65+, 2D4+ 65-, 2D4- 65+, and 2D4- 65-. Functional T cell clones in all four subfamilies have been observed. Cytolytic function can be correlated with the TcR phenotype expressed because all of the Mab which react with a particular clone inhibit its ability to lyse a specific target. The epitopes recognized by the panel are closely related because all five block each other's binding to HPB-ALL. In addition, the determinants recognized by 3D6, 1C1, and 1C2 on normal lymphocytes are probably very closely related because all clones examined react with all three Mab.     

CA215 and GnRH receptor as targets for cancer therapy

Two monoclonal antibodies (Mabs), RP215 and GHR106, were selected for the preclinical evaluations of anti-cancer drugs targeting various human cancers including those of the ovary, cervix, lung, and liver. Both Mabs were shown to react with pan cancer markers, which are over-expressed on the surface of almost all human cancers. RP215 Mab was shown to react with the carbohydrate-associated epitope(s) of cancer cell-expressed glycoproteins, mainly consisting of immunoglobulin superfamily (IgSF) proteins and mucins, generally known as CA215. GHR106 Mab was generated against the extracellular domain of human GnRH receptor, which is also highly expressed on the cancer cell surface. Preclinical studies were performed to evaluate the efficacy of these two Mabs as anti-cancer drugs for treating human cancers. High tumor specificity of RP215 Mab was demonstrated with immunohistochemical staining studies of various cancer cell lines, as well as normal and cancerous tissue sections. These two Mabs were shown to induce apoptosis as well as complement-dependent cytotoxicity upon treatment to many cultured cancer cells. Significant dose-dependent growth inhibition of tumor cells from several different tissue origins were demonstrated by nude mouse experiments. It was further demonstrated that GHR106 Mab can function as long-acting GnRH analogs in its biological actions. Efforts were made to generate human/mouse chimeric forms of the GHR106 Mab. Based on the results of these preclinical studies, we believe that these two Mabs, in chimeric or humanized forms, can be developed into suitable therapeutic agents for treatment of human cancers as anti-cancer drugs.     

Monoclonal antibodies to the 27-34K insulin-like growth factor binding protein

Monoclonal antibodies were prepared against the 27-34K insulin-like growth factor (IGF)-binding protein purified from human placenta/decidua and designated placental protein 12 (PP12). Four different antibodies were characterized. Each recognized the major band at 32K on immunoblots of the purified PP12 preparation and amniotic fluid. In liquid phase RIA, IGF-I did not affect the binding of [125I] PP12 to one antibody (Mab 6303), it slightly increased the binding to two antibodies (Mab 6301 and 6304), and it slightly decreased the binding to one antibody (Mab 6302). All antibodies immunoprecipitated the cross-linked PP12-[125I] IGF-I complex, but Mab 6302 considerably less effectively than the others. Preincubation of PP12 with Mab 6302 completely inhibited the binding of [125I] IGF-I to PP12, whereas preincubation with Mab 6303 had no effect, and Mab 6301 as well as Mab 6304 increased it. These results suggest that Mab 6302 binds to an epitope at or near to the IGF-binding site, whereas the other antibodies react at other sites of the PP12 molecule. Conformational changes in PP12 probably account for the IGF-I-induced increase in the binding of Mabs 6301 and 6304 to [125I] PP12, and vice versa, for Mabs 6301- and 6304-induced increase in the binding of [125I] IGF-I to PP12.     

Primary sequence mapping of human apolipoprotein B-100 epitopes. Comparisons of trypsin accessibility and immunoreactivity and implication for apoB conformation

Differential trypsin-accessibility and monoclonal antibodies (Mabs) to human apolipoprotein (apo) B-100 are both important tools for probing apoB structure and conformation on low-density lipoproteins (LDL). In this study, we have mapped greater than 80% of the C-terminal region (720 residues) of LDL apoB-100 using trypsin digestion. Our results extend our previous data [Yang et al. (1986) Nature (Lond.) 323, 738-742] confirming that the C-terminal region of about 420 residues of apoB-100 is largely inaccessible to trypsin, whereas the part just preceding this region has interspersed trypsin-accessible and inaccessible peptides. We have determined the amino acid sequence of specific apoB-100 peptides containing epitopes recognized by four separate Mabs: two epitopes have been mapped to within 20 residues, one has been mapped to 36 residues, and the last to 80 residues. We used polyclonal antisera to identify 16 overlapping clones of varying lengths of apoB-100 cDNAs extending from the C-terminus of apoB-100 cloned in the expression vector, lambda gt11. These clones were then tested against individual Mabs. By nucleotide sequence analysis of overlapping clones that show differential reactivities to different Mabs, we have mapped the individual epitopes of each Mab to within about 50-150 amino acid residues predicted from the DNA sequences. Confirmation and further fine mapping were accomplished by competition for LDL binding using partially purified fusion proteins and chemically synthesized oligopeptides. Two epitopes (Mabs 7 and 22) were mapped to the C-terminal 20 amino acids of apoB-100, one (Mab 16) to residues 4154-4189, and another (Mab 20) to residues 3926-4005. Mab 16 precipitates more than 80% of LDL particles. Mab 20 precipitates only denatured apoB but not native LDL apoB [Milne et al. (1987) Mol. Immunol. 24, 435]. Mabs 7 and 22 are unique in that they precipitate LDL apoB modified by storage much better than freshly isolated LDL-apoB. Although epitope expression and trypsin-accessibility represent two useful probes for the study of protein conformation, there was no obvious correlation between these two parameters when applied to LDL apoB for the antibodies we have examined.     

A precursor form of latent collagenase produced in a cell-free system with mRNA from rabbit synovial cells

mRNA extracted from rabbit synovial fibroblasts which had been induced to produce large amounts of collagenase (EC 3.4.23.7) by urate crystals was translated in a cell-free wheat germ system. Collagenase was identified by immunoprecipitation using mono-specific antibody to rabbit synovial collagenase. In the absence of microsomal membranes, a single precursor with Mr = 59,000 was synthesized. This polypeptide was susceptible to proteolytic degradation. In the presence of canine pancreatic microsomes, the nascent protein was processed to a polypeptide with Mr = 57,000 (identical in mobility on sodium dodecyl sulfate-gel electrophoresis to the major latent collagenase secreted from cells) and was protected from tryptic digestion unless a detergent was used to disrupt the membranes. In addition to Mr = 57,000 material, cells secreted immunologically reactive latent collagenase with Mr = 61,000. High molecular weight collagenase was separated from Mr = 57,000 species by binding to concanavalin a-Sepharose, suggesting that this enzyme was a product of post-translational glycosylation. Both latent enzymes were activated by trypsin and human plasma kallikrein to Mr = 45,000 and 49,000. The evidence indicates that rabbit synovial fibroblast collagenase is synthesized and secreted as a single polypeptide zymogen, not as an enzyme-inhibitor complex.     

Sulfated oviductal glycoproteins in the rabbit: quantitation by competitive enzyme-linked immunosorbent assay

The rabbit oviductal epithelium synthesizes and secretes a family of antigenically related, sulfated oviductal glycoproteins (SOG). Anti-SOG monoclonal antibodies (Mabs) were produced and two (Mab 1 and Mab 2) were selected for further characterization. Periodate oxidation of Western blots of oviductal fluid did not affect the binding of Mab 1 or Mab 2, thus suggesting that these antibodies recognized protein rather than carbohydrate epitopes on SOG. The specificity of Mab 1 was determined by Western blot analysis of tissues obtained from estrous rabbits and from the male rabbit reproductive tract. SOG was identified in tissue extracts of both the oviductal ampulla and isthmus. Cervix was the only non-oviductal tissue with which Mab 1 cross-reacted. Mab 1 was used to isolated SOG from whole oviductal fluid by immuno-affinity chromatography. Affinity-purified SOG and Mab 1 were used to develop a quantitative, SOG-specific, competitive enzyme-linked immunosorbent assay. This assay was used to quantify SOG in rabbit oviductal fluid collected during estrus and pseudopregnancy. SOG secretion during pseudopregnancy was resolved into two transient episodes of increased secretion. Maximum SOG secretion (X = 1039 +/- 199 micrograms/day) occurred within 48 h of the induction of pseudopregnancy. A second period of enhanced SOG secretion (X = 308 +/- 46 micrograms/day) occurred during the fifth and sixth days of pseudopregnancy. Baseline SOG secretion occurred during estrus at approximately 60% of maximum postovulatory secretion.     

Production of procollagenase by cultured human keratinocytes

Using a collagen film assay utilizing 14C-labeled type I collagen, we demonstrated that cultured human keratinocytes produced a procollagenase after treatment with the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Production of collagenase paralleled alterations in cellular morphology induced by TPA. When procollagenase was immunoprecipitated with antibody to human fibroblast collagenase and analyzed on sodium dodecyl sulfate-polyacrylamide gels, the zymogen was revealed as a 56- and 51-kDa doublet. The keratinocyte-derived collagenase was a neutral metalloprotease, required activation with trypsin for detection in the collagenase assay and produced the characteristic three-quarter and one-quarter length collagen cleavage products when incubated with type I collagen at 25 degrees C. The enzyme was inhibited by serum and cysteine and was largely unaffected by serine, thiol, and carboxyl protease inhibitors. Cycloheximide inhibited the TPA-induced production of collagenase, suggesting that the procollagenase was not stored preformed in the keratinocytes. Keratinocytes treated with a tumor-promoting analogue of TPA also produced collagenase, but cells treated with cytochalasin B, interleukin-1, or two non-tumor promoting phorbol esters did not. Keratinocyte-derived collagenase may play a role in wound healing and morphogenesis.     

Activation of acetate by Methanosarcina thermophila. Purification and characterization of phosphotransacetylase

Phosphotransacetylase (EC 2.3.1.8) was purified 83-fold to a specific activity of 2.5 mmol of acetyl-CoA synthesized per min/mg of protein from Methanosarcina thermophila cultivated on acetate. This rate was 10-fold greater than the rate of acetyl phosphate synthesis. The native enzyme (Mr 42,000-52,000) was a monomer and was not integral to the membrane. Activity was optimum at pH 7.0, and 35-45 degrees C. The enzyme was stable to air and to temperatures up to 70 degrees C, but was inactivated at higher temperatures. Phosphate and sulfate partially protected against heat inactivation. Potassium or ammonium ion concentrations above 10 mM were required for maximum activity of the purified enzyme; the intracellular potassium concentration of M. thermophila approximated 175 mM. Sodium, phosphate, sulfate, and arsenate ions were inhibitory to enzyme activity. Western blots of cell extracts showed that phosphotransacetylase was synthesized in higher quantity in acetate-grown cells than in methanol-grown cells.     

The development of porcine zona pellucida using monoclonal antibodies: I. Immunochemistry and light microscopy

We established three monoclonal antibodies (Mabs) against the zonae pellucidae (ZP) of porcine oocytes, named STA-1, STA-2, and STA-3, and eventually we determined that they all reacted with the isolated ZP. Based on Western blotting without 2-mercaptoethanol (2-ME), STA-1 reacted with the 80,000-110,000 Mr component, STA-2 with the 42,000-63,000 Mr component, and STA-3 with the 40,000-80,000 Mr component of ZP. We immunohistochemically specified the components of porcine ZP reactive with the three Mabs during the course of follicular development. Each Mab reacted with both the ZP and the interfollicular cell space (IFCS). One ZP component, reactive with STA-2 and STA-3, was first produced in the primordial follicle and was not found at the cumulus follicle stage, which corresponds to the stage of large antral follicles more than 5 mm in diameter. Another ZP component, reactive with STA-1, was not produced until the secondary follicle stage, and was never found at the antral follicle stage. These results suggest that each ZP component is produced and secreted at a specific stage or stages of folliculogenesis.     

Characterization of multi-cellulases of Sporotrichum cellulophilum by using monoclonal antibodies

Cellulases of Sporotrichum cellulophilum, CII1, CII2, CII3, CII5, CIII1, CIII2, and CIV1 were purified to homogeneity by column chromatographies and preparative electrophoresis. The specific activity of CII3 was the highest (100 units/mg), while the activities of CII5, CIII1, and CII2 were low (12–17 units/mg). The molecular weight of CII2 was 19,000, while those of the others ranged from 46,000–63,000. All the cellulases were glycoproteins except for CII2. Three monoclonal antibodies (Mab CII11, Mab CII12, and MabCII13) were prepared against CII1, and Mab CIV11 against CIV1. From the immunological reactivities, it was found that CII1, CIII2, and CIV1 had a common antigenic site recognized by Mab CII11, Mab CII12, and Mab CII13, while CII12 and CVI1 had a different antigenic site that was recognized by Mab CIV11, MabCII11, MabCII12, MabCII13, and MabCIV11 did not react with CII2, CII3, CII5, and CIII1. The classification of these cellulases was discussed.     

K562 erythroleukemia cells express cytokeratins 8, 18, and 19 and epithelial membrane antigen that disappear after induced differentiation.

The effects of differentiation-modulating drugs were studied on the expression of intermediate filaments (IFs) in the human K562 erythroleukemic cell line. The untreated cells contained typical cytoplasmic coiling bundles, positive for both vimentin and cytokeratin as judged by indirect immunofluorescence microscopy with monoclonal antibodies (Mabs). Some of the cells also showed bright immunoreactivity for epithelial membrane antigen (EMA), as revealed with a Mab and polyclonal antiserum. When exposed to hemin or to sodium butyrate, most of the cells became cytokeratin negative within 3 days and showed dispersion of vimentin fibrils. Upon exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the amount of both vimentin and cytokeratin appeared to be greatly increased within 3 days and was found both in dispersed cytoplasmic fibrils, in large spherical, eccentric aggregates, as well as in cytoplasmic fibrils in cells spreading on fibronectin. TPA induced a complete loss of proliferation, as judged by immunostaining with the Mab Ki-67. The effects of TPA were found to be irreversible and could be induced by only a short exposure to the drug. Western blotting analysis and monoclonal antibodies to individual cytokeratins revealed that untreated K562 cells expressed Mr 52,000 (No. 8), 46,000 (No. 18), and 40,000 (No. 19) cytokeratin polypeptides, which disappeared when the cells were exposed to hemin or to sodium butyrate to induce erythroid differentiation but were greatly enhanced when exposed to TPA. The monoclonal anti EMA antibody reacted in K562 cells with a single Mr 320,000 polypeptide that was also revealed in MCF-7 breast carcinoma cells. Human bone marrow cells or other leukemic cell lines with erythroid differentiation capacity (HEL and KG-1) did not contain cytokeratin- or EMA-immunoreactive cells, suggesting that in K562 cells these properties may rather represent abnormal cytodifferentiation or retrodifferentiation toward early embryonic mesenchymal cells, than a more general expression of epithelial features in human leukemic cells.     

Monoclonal antibodies to the bovine immunoglobulin G2a allotypes A1 and A2.

Production of murine monoclonal antibodies (Mabs) to bovine IgG2a-A1 and A2 allotypes resulted in three Mabs being selected as anti-IgG2a-A1 and A2 reagents. Two Mabs recognize the A1 allotype and 1 recognizes the A2 allotype. Initial epitope mapping with the Mabs indicates that one of the A1 epitopes resides in the hinge region and the other epitope resides more toward the C-terminus of the immunoglobulin. The A2 epitope recognized by the A2 Mab does not appear to reside in the hinge region of the immunoglobulin but apparently resides more C-terminal.