Monoclonal antihuman thyroglobulin antibodies

Six murine hybridomas secreting monoclonal antihuman thyroglobulin (Tg) antibodies (TAK 1-6) were established by cell fusion techniques. Solid phase RIA was employed to detect the anti-Tg antibody in culture supernatants of hybridomas. The characteristics of these monoclonal antibodies were analyzed by radioimmune blocking assay using rat Tg, human glycoproteins, thyroid hormones, and various preparations of Tg obtained from patients with thyroid disease as inhibitors. In the same system, competitive inhibition studies between 125I-labeled and unlabeled monoclonal antibodies were carried out to determine whether these antibodies recognized the same antigenic determinant. TAK 2 and 3 reacted with human Tg specifically and had equal binding activity using various preparations of human Tg. The other four monoclonal antibodies (TAK 1, 4, 5, and 6) cross-reacted with xenogeneic Tg (rat Tg) and their affinity for human Tg increased as the iodine content of Tg increased. Tg binding to TAK 1 and 4 was inhibited by T4, whereas Tg binding to TAK 5 and 6 was not inhibited by any thyroid hormone or their precursors. In conclusion, we prepared six monoclonal antihuman Tg antibodies. One group is specific for human Tg and recognizes the framework structure unmodified by iodination; the second group reacts with iodination-related epitopes other than iodoamino acids, and the third group recognizes determinants consisting of T4. These monoclonal antibodies provide important probes to detect the polymorphism of human Tg.     

Monoclonal antibodies to human prostatic acid phosphatase: probes for antigenic study.

Hybrid cell lines producing monoclonal antibodies against human prostatic acid phosphatase [PAPase; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) were prepared by the fusion of mouse myeloma cells with the spleen cells of PAPase-immunized BALB/c mice. Approximately 23% of the hybrid cells initially plated after cell fusion produced specific antibodies: 34 microcultures were cloned, and 8 eventually yielded stable cell lines. The monoclonal antibodies produced by these eight hybridomas were characterized for their isotypes, isoelectric points, concentrations, and affinities. All of the eight monoclonal antibodies exhibited strict specificity for PAPase as determined by radioimmunoassay and immunohistochemical methods. These antibodies were used as probes for the antigenic mapping of this enzyme, and three nonoverlapping determinants were recognized. Further binding studies with PAPase fragments, generated by cleavage with a submaxillaris protease, showed that those three determinants are clustered on one fragment of PAPase. These monoclonal antibodies may be useful in refinement of clinical immunoassays of PAPase or immunohistological study of PAPase-synthesizing cells.     

Monoclonal antibodies as probes to study the human growth hormone-binding domain to lactogenic rat liver receptors.

A set of monoclonal antibodies (MAb) to human GH (hGH) was used to study the hormone binding orientation to its receptors (R) from female rat liver. The hGH antigenic region left exposed after its binding to liver microsomes was detected by measuring the ability of various [125I]MAb to bind to the preformed hGH-R complexes. Results indicated that a cluster of epitopes defined by the MAb, termed AE5, AC8, and AE12, remains accessible in the hGH-R complex whereas overlapping epitopes 3C11 and HG3 would define a hGH region involved in the binding site. Supporting these findings, solubilization and HPLC gel filtration of [125I]MAb-hGH-R complexes showed a radioactive peak of about 450,000 mol wt for MAb AE5 or AC8, but not for MAb 3C11 or HG3. [125I]MAb AE12 behaved differently, suggesting that epitope AE12 may be masked or altered in hGH-R-solubilized complexes. MAb directed to the putative hGH-binding site (MAb 3C11, HG3, and the closely related MAb 10C1 and NA71) failed to inhibit binding of the preformed [125I]MAb AE5-hGH complex to the receptors, suggesting a hormone modification after MAb AE5 binding. Accordingly competition experiments indicated an increase in the affinity of hGH for its receptors induced by this MAb. A higher hGH concentration was required to obtain 50% [125I]hGH binding to liver microsomes in the presence of MAb AE5 than in its absence. As the MAb used define epitopes that were previously correlated with the hGH structure, we concluded that a high flexible region (sequences 134-150) is exposed in the hGH-R complex. Furthermore, some MAb directed to this region enhance the hormone affinity for its rat liver receptors, probably through an induced conformational change.     

Monoclonal antibodies to thyroglobulin elucidate differences in protein structure of thyroglobulin in healthy individuals and those with papillary adenocarcinoma

Monoclonal antibodies specific for human thyroglobulin (Tg) from normal subjects were prepared by the hybridoma technique. Antibodies from three clones (clones B2F, C6E, and C6G) were found to produce linear Scatchard plots, as predicted for homogeneous antibodies. Based on different patterns of cross-reactivity with Tg from various species, these monoclonal antibodies recognized different determinants on the Tg molecule. Moreover, antibodies from clone B2F bound simultaneously with clone C6E or C6G to Tg. Therefore, antibody from clone B2F must bind to a site on Tg distant from those recognized by clone C6E or C6G. The monoclonal antibodies C6G and C6E bound almost equally to normal Tg and Tg from patients with Graves' disease, adenoma, follicular carcinoma, and papillary adenocarcinoma. In contrast, whereas clone B2F bound equally well to normal Tg and Tg from patients with Graves' disease, adenoma, and follicular carcinoma, this clone bound poorly to Tg from patients with papillary adenocarcinoma. Since the binding activity of clone B2F for unfolded or degenerated Tg was remarkably decreased, these differences in binding activities to native Tg may reflect changes in conformation of the Tg molecule. Thus, the results indicate there may be conformational changes in Tg from patients with different thyroid diseases.     

Monoclonal antibodies directed to human and equine chorionic gonadotropins as probes for the topographic analysis of epitopes on the human alpha-subunit

To improve our knowledge of the structural features of the alpha-subunit of hCG we have studied the antigenic site recognized by monoclonal antibody (MAb) ECG01 raised against equine CG (eCG) which binds to hormones and alpha-subunits from human and equine species. We have also delineated regions of hCG alpha comprising the epitope recognized by HT13 which was raised against hCG and binds to hCG and hCG alpha. To define the residues involved in the antigenic sites recognized by ECG01 and HT13, we have studied the reactivities of these two MAbs with native or chemically modified LH and CG with subunits from equine, human, or ovine (o) species or with synthetic peptides analogous to various portions of hCG alpha. We have also compared these reactivities with those displayed by MAbs AHT20 and FA 36, whose epitopes have been previously described; anti-hCG alpha MAb AHT20 is specific for the free alpha-subunits of various species and recognizes residues localized to the 36-41 region of hCG alpha, whereas antipeptide MAb FA36 binds to the 87-92 carboxyl-terminal part of hCG alpha. Our results show that the epitopes of HT13 and ECG01 are 1) probably discontinuous, as these MAbs did not bind to the reduced and S-carboxymethylated hCG alpha; and 2) constituted by residues borne on the 1-35 and 52-86 sequences, as they do recognize the hCG alpha core missing the 36-51 portion, yet do not recognize hCG alpha-(87-92) region recognized by FA36. The comparative studies performed with specific two-site immunoradiometric assays to determine the interspecies cross-reactivities of the MAbs allow us hypothetical assignment of residues on the primary structure of hCG alpha. The antigenic site recognized by ECG01 might include two to six amino acids, four of these residues being located at inverted places compared to those of oLH alpha (Asp6/Gly22 and Arg67/Lys75). These residues present important charged functional groups highly conserved among evolutionarily related variants, and it is likely that they are located on the surface of both the intact hormone and its alpha-subunit. Three peptidic portions of hCG alpha, 16-17, 64-66, and 73-76, respectively, might be involved in the epitope recognized by HT13, although we could not rule out the possibility that other residues were also involved in the antigenic site. These observations allow us to identify several residues as potentially constituting the epitopes recognized by two MAbs on both hCG and hCG alpha.     

Monoclonal antibodies as molecular probes to study structural heterogeneity between human and animal renins and other aspartyl proteinases

Monoclonal antibodies may be helpful in comparing the structural homology of renins of various animal species as well as other aspartyl proteinases. We obtained four hybridoma lines secreting monoclonal antibodies that inhibited 3 Goldblatt mU human renin enzymatic activity with apparent IC50 of 1.1 X 10(-6)-4 X 10(-8) M. Each antibody was specific for human renin and did not cross-react either with other proteins tested nor with renins of other species in an immunoradiometric assay. These antibodies did not inhibit the enzymatic activities of nonprimate renins or nonrenin aspartyl proteinases tested. These data indicate that certain structural determinants of human renin activity may be uniquely different from those of nonprimate renins or nonrenin aspartyl proteinases.     

Monoclonal antibodies to human intrinsic factor

Mice were immunized with human intrinsic factor, and their lymph node cells were fused with a myeloma cell line by standard hybridoma techniques. Eleven of the resulting 227 hybridomas secreted immunoglobulin G capable of binding to intrinsic factor-cobalamin complex. Cloning by limiting dilution gave 6 clones secreting anti-intrinsic factor antibodies that bound human intrinsic factor-cobalamin complex with affinities of 13-116 nM; 3 antibodies also bound rabbit intrinsic factor-cobalamin complex. Five antibodies inhibited to some degree the binding of cobalamin by intrinsic factor, and 2 also prevented attachment of intrinsic factor-cobalamin complex to guinea pig ileal receptors. Anti-rabbit intrinsic factor antibodies specifically precipitated a peptide of molecular weight 53,000, corresponding to the molecular weight of rabbit intrinsic factor from homogenates of rabbit gastric mucosal explants biosynthetically labeled with [35S]methionine and from culture medium in which the explants were incubated. Indirect fluorescence immunocytochemistry with the antibodies in human and rabbit gastric mucosal sections showed intense selective staining of parietal cells. These results (a) document species differences between human and rabbit intrinsic factors not previously demonstrable with polyclonal anti-intrinsic factor sera; (b) confirm earlier evidence that cobalamin binding and receptor functions occur at separate sites in intrinsic factor; and (c) provide a useful approach to studying structure-function relations of the intrinsic function molecule.     

Monoclonal antibodies to human pancreatic carcinoma

Hybridoma cultures were produced by the fusion of SP2 mouse myeloma cells with spleen cells from mice immunized with human pancreatic carcinoma cells. After limiting dilutions, three monoclonal antibodies, YPan1, YPan2, and YPan3, which bound to immunizing cells but not to normal human skin fibroblasts, were further characterized. The three monoclonal antibodies were found to bind to all seven pancreatic carcinoma cell lines but not to other carcinoma cell lines tested except some colon carcinoma cell lines. When human tissue sections were examined using immunohistochemical techniques, the three monoclonal antibodies identified antigens in the pancreatic carcinomas and some normal pancreases, but only YPan1 showed strong positive staining. No cross-reactivity was seen in sections of other carcinomas tested except some colon carcinomas. The results suggest that these monoclonal antibodies may be usefully applied to the detection of pancreatic carcinomas.     

Monoclonal antibodies to estrophilin: probes for the study of estrogen receptors.

Splenic lymphocytes from a Lewis rat, immunized with purified estradiol-receptor complex of calf uterine nuclei, were fused with cells of three different mouse myeloma lines (P3-X63-Ag8, P3-NSI/I-Ag4-1, and Sp2/0-Ag14) to yield hybridoma cultures, 9% of which produced antibodies to the receptor protein (estrophilin). When cloned by limiting dilution, approximately 70% of the viable cultures secreted antiestrophilin antibody. When expanded in suspension culture, three clones derived from Sp2/0-Ag14 were found to secrete rat IgG (gamma 2a class), whereas seven other clones (from all three myeloma lines) secreted IgM. Monoclonal IgG shows comparable affinity for nuclear and extranuclear receptors, whereas IgM reacts preferentially with the nuclear form. Both classes of antibody react with unoccupied as well as with occupied receptor and do not interfere with its ability to bind to estradiol. By growing IgG-secreting clones in the presence of [35S]methionine, radiolabeled monoclonal antiestrophilin has been prepared. Unlike antiestrophilin antibody previously generated in the rabbit or the goat, which crossreacts with estrogen receptors from every animal species tested, antibodies produced by the Lewis rat and by hybridomas derived from its spleen cells react specifically with estrophilin from calf tissues. These monoclonal antibodies provide reagents for the application of immunochemical techniques to study estrogen receptors in calf target tissues.     

Monoclonal antibodies as probes for unique antigens in secretory cells of mixed exocrine organs.

In the past, it has been difficult to identify the secretory product and control mechanisms associated with individual cell types making up mixed exocrine organs. This report establishes the feasibility of using immunological methods to characterize both the biochemical constituents and regulatory mechanisms associated with secretory cells in the trachea. Monoclonal antibodies directed against components of tracheal mucus were produced by immunizing mice with dialyzed, desiccated secretions harvested from tracheal organ culture. An immunofluorescence assay revealed that of the total 337 hybridomas screened, 100 produced antibodies recognizing goblet cell granules; 64, gland cell granules; and 3, antigen confined to the ciliated apical surface of the epithelium. The tracheal goblet cell antibody described in this report was strongly cross-reactive with intestinal goblet cells, as well as with a subpopulation of submandibular gland cells, but not with cells of Brunner's glands or the ciliated cell apical membrane. The serous cell antibody was not cross-reactive with goblet, Brunner's gland, or submandibular cells, or the ciliated cell apical membrane. The antibody directed against the apical membrane of ciliated cells did not cross-react with gland or goblet cells or the apical membrane of epithelial cells in the duodenum. Monoclonal antibodies, therefore, represent probes by which products unique to specific cells or parts of cells in the trachea can be distinguished. The antibodies, when used in enzyme immunoassays, can be used to quantitatively monitor secretion by individual cell types under a variety of physiological and pathological conditions. They also provide the means for purification and characterization of cell-specific products by immunoaffinity chromatography.     

Monoclonal antibodies to human estrogen receptor.

Extranuclear estrogen receptor protein (estrophilin) of MCF-7 human breast cancer cells was purified by passage of the cytosol fraction of a cell homogenate through an affinity column of estradiol linked to Sepharose by a substituted di-n-propyl sulfide bridge in the 17 alpha position. Elution with 50 micro M [3H]estradiol in 10% (vol/vol) dimethyl formamide/0.5 M sodium thiocyanate gave 40% recovery of [3H]estradiol-estrophilin showing 14% of the specific radioactivity expected for the pure complex. Serum from a Lewis rat immunized with this partially purified estradiol-receptor complex contained antiestrophilin antibodies that reacted not only with nuclear and extranuclear estradiol-receptor complexes from MCF-7 cells but also with estrophilin from rat, calf, and monkey uterus, hen oviduct, and human breast cancers. Splenic lymphocytes from the immunized rat were fused with cells of two different mouse myeloma lines (P3-X63-Ag8 and Sp2/0-Ag14) to yield hybridoma cultures, 2% of which produced antibodies to estrophilin. After cloning by limiting dilution, three hybridoma lines secreting antiestrophilin were expanded in suspension culture and as ascites tumors in athymic mice to provide substantial quantities of monoclonal antibodies that recognize mammalian but not avian estrophilin and that show different degrees of reactivity with receptor from nonprimate sources. By growing the clone from Sp2/0 in the presence of [35S]methionine, radiolabeled monoclonal IgG has been prepared. These monoclonal antibodies should prove useful in the study of estrogen receptors of human reproductive tissues, in particular for the radioimmunochemical assay and immunocytochemical localization of receptors in breast cancers.     

Monoclonal antibodies to nucleic acid-containing cellular constituents: probes for molecular biology and autoimmune disease

Mice of the strain MRL/Mp-lpr/lpr develop a lupus erythematosus-like syndrome that includes the production of autoantibodies specific for nucleic acid-containing cellular components. We have fused spleen cells from such a mouse with the myeloma SP 2/0 and examined the antibodies produced by the resultant cloned hybrid cell lines by using immunoprecipitation and immunofluorescence techniques. Three types of monoclonal antibodies, specific for Sm, DNA, or rRNA, all antigens to which patients who have lupus make antibodies, have been identified. Patient anti-Sm antibody had previously been reported to precipitate five small nuclear ribonucleoproteins that contain U-1, U-2, U-4, U-5, and U-6 RNAs. The monoclonal anti-Sm antibody gives the same immunoprecipitation pattern, providing direct evidence that the Sm antigen resides on all these RNA-protein complexes. Monoclonal anti-Sm antibody will be valuable in deciphering the biological function of these ubiquitous small nuclear RNPs. A simple competition radioimmunoassay using the monoclonal anti-Sm antibody to titer patient sera is also presented. Uses of monoclonal antibodies for the study of autoimmune disease are discussed.     

Monoclonal antibodies to human vitamin K-dependent protein S

Monoclonal antibodies to human protein S have been prepared using established hybridoma technology. One antibody was isolated that binds protein S only when Ca2+ is present; others bind antigen equally well in the presence or absence of EDTA. Other antibodies display a diminished affinity for protein S in the presence of EDTA. Purified immunoglobulins from cell lines displaying Ca2+ dependence were immobilized and used to purify protein S from fractions obtained by barium precipitation of citrated plasma, ammonium sulfate fractionation, and chromatography on diethylaminoethanol (DEAE)- Sephadex and dextran sulfate agarose. Essentially homogeneous protein S was isolated from the barium-citrate-adsorbed, 35% ammonium-sulfate- soluble proteins using a totally Ca2+-dependent antibody and EDTA elution. Protein S and several substances of higher mol wt were bound directly from plasma by a partially Ca2+-dependent antibody and were eluted partially with EDTA and NaCl and finally with NaSCN. The largest and most abundant of the high mol wt materials is likely protein S- complement C4b-binding protein complex. The immunoaffinity-isolated protein S was found to be indistinguishable from conventionally isolated protein S in terms of activity, sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) mobility, and by high- performance liquid chromatography (HPLC). These studies establish reagents that can probe the structure of protein S and isolate protein S in its free and complexed forms.     

Monoclonal antibodies to human vitamin D-binding protein.

Monoclonal antibodies to vitamin D-binding protein isolated from human serum have been produced. The antibodies obtained have been shown to be specific for human vitamin D-binding protein by three independent assays. The antibodies recognize human vitamin D-binding protein specifically in an enzyme-linked immunosorbent assay. Human vitamin D-binding protein is detected specifically in both pure and crude samples by a radiometric immunosorbent assay (RISA) and by an immunoprecipitation assay. The anti-human vitamin D-binding protein antibodies cross-react with monkey and pig vitamin D-binding protein, but not with vitamin D-binding protein from rat, mouse, or chicken, as determined by the RISA and immunoprecipitation assays.     

Monoclonal antibodies against human follicle-stimulating hormone

Five monoclonal antibodies to human (h) FSH have been prepared, isolated, and characterized. They were produced by hybridomas derived from FO myeloma cells and spleen cells from mice immunized with hFSH or its beta-subunit (hFSH beta). Two of the antibodies (A101 and A102) which recognized the alpha-subunit of hFSH bound much better when alpha was associated with the beta-subunit forming the intact hFSH molecule than when alpha was in free form. These antibodies showed 9-10%, 2-3%, and 1-3% cross-reactions with alpha-subunits in hTSH, hCG, and hLH, respectively. Two antibodies (B201 and B202) recognized only free beta-subunit. One antibody (B305) recognized free beta-subunit and native hFSH. There was no significant cross-reaction of these antibodies to FSH beta with hTSH, hLH, and hCG. Using solid phase competitive binding and sandwich assays, we compared the epitopes for these antibodies. Antibodies A101 and A102 recognize the same epitope on hFSH alpha. Antibodies B201 and B202 recognize different epitopes, but they seemed to be adjacent. Antibody B305 bound a different epitope than B201 and B202. Such characteristics of these antibodies can be useful for sensitive and specific assay of hFSH or hFSH beta and also may be helpful in studying FSH interaction with its receptor.     

Monoclonal antibodies to human coagulation factor V and factor Va

BALB/c mice were immunized with human factor V. The immunogen was a mixture of procofactor (factor V) and thrombin-activated cofactor (factor Va). Spleen cells were obtained from an immunized animal and fused with NS-1 murine myeloma cells. Hybrid cell cultures were assayed for the production of antibodies to human factor V and factor Va by a solid-phase radioimmunoassay. Factor V and/or factor-Va-specific antibodies were detected in 38 of the 96 cultures assayed. The cells from 10 of these positive cultures were subcloned by limiting dilution and grown as ascites tumors in BALB/c mice. Ascitic fluids were obtained and characterized with respect to their binding interaction with human factor V and factor Va. Three hybridoma cell lines produce monoclonal antibodies that react equally well with factor V and factor Va. Another antibody reacts with both antigens, but the reactivity with factor V is better than with factor Va. An additional two antibodies react with factor Va better than factor V in the radioimmunoassay (RIA). The remaining four antibodies react exclusively with factor V. A previously described murine monoclonal antibody to human factor V (alpha HFV-1) has been used to study the peptides produced during the thrombin-catalyzed activation of human factor V. This antibody binds both factor V and factor Va, releases them at high ionic strength, and has an apparent dissociation constant for factor Va of 3 x 10(-9)M. When human factor V (mol wt 330,000) is activated by thrombin and passed over an alpha HFV-1-Sepharose affinity resin, factor Va binds and subsequently can be eluted. The eluate in 1.2 M NaCl contains two fragments of apparent mol wt 93,000 and 70,000. EDTA, which inactivates factor Va, promotes release of the mol wt 93,000 fragment from factor Va bound to the antibody. Subsequent elution with 1.2 M NaCl releases the mol wt 70,000 fragment. These observations indicate that human factor Va is a two subunit protein and that the epitope for alpha HFV-1 is on the mol wt 70,000 fragment.     

Monoclonal antibodies to bovine prolactin interacting with human prolactin]

Two stable hybridomas producing antibodies (Mab 1 and Mab 2) to bovine prolactin and belonging to the IgG1 subclass have been prepared. The cross-reactivity of Mab 1 and Mab 2 with some structurally similar pituitary protein (human, pig, whale, rat prolactins, bovine and human somatotropins) using indirect immunoenzymatic assay, was studied. It has been shown that Mab 2 reacts specifically only with bovine prolactin whereas Mab 1 interacts with human prolactin and prolactins of different animals. The specificity of Mab 1 to human prolactin was confirmed by immunoradiodetection assay on nitrocellulose filters. The data obtained give evidence of the existence of at least two different sterically nonoverlapping epitopes: one of them is specific exclusively for bovine prolactin and the other one is common, i.e. extraspecific.