Immunoperoxidase staining of carcinoembryonic antigen with monoclonal antibodies in adenocarcinoma of the colon

Mouse monoclonal antibodies to carcinoembryogenic antigen (CEA) obtained by the somatic cell hybridization technique of K?hler and Milstein were used in a modified enzyme bridge immunoperoxidase staining method. Both high and low affinity antibodies were tested and their staining properties compared with those of a commercial polyvalent rabbit antiserum. The staining pattern of neoplastic epithelial cells in all seven antibodies in samples of primary adenocarcinoma of the colon was similar, indicating that no gross differences were found in the exposure of the different antigenic determinants of CEA in formalin fixed tissue. The background staining of the monoclonal antibodies are negligible. It is concluded that monoclonal antibodies are superior to conventional antisera in immunoperoxidase staining of CEA.     

Immunoperoxidase staining of carcinoembryonic antigen with monoclonal antibodies in adenocarcinoma of the colon

Summary Mouse monoclonal antibodies to carcinoembryogenic antigen (CEA) obtained by the somatic cell hybridization technique of Köhler and Milstein were used in a modified enzyme bridge immunoperoxidase staining method. Both high and low affinity antibodies were tested and their staining properties compared with those of a commercial polyvalent rabbit antiserum. The staining pattern of neoplastic epithelial cells in all seven antibodies in samples of primary adenocarcinoma of the colon was similar, indicating that no gross differences were found in the exposure of the different antigenic determinants of CEA in formalin fixed tissue. The background staining of the monoclonal antibodies was negligible. It is concluded that monoclonal antibodies are superior to conventional antisera in immunoperoxidase staining of CEA.     

Subcellular localization of glycogen synthase with monoclonal antibodies

Two monoclonal antibodies, designated 7H5 and 8E11, were produced against glycogen synthase purified from rabbit skeletal muscle. Both antibodies were of the IgG1 (k) isotype. Western blot analysis of extracts of rat and rabbit tissues showed that antibody 7H5 recognized glycogen synthase from skeletal and cardiac muscles, but not from liver. Antibody 8E11 gave similar results but the responses were weaker. Antibody 7H5 also recognized a 69,000 dalton tryptic fragment of glycogen synthase whereas antibody 8E11 did not bind this fragment. Immunocytochemical staining of rabbit skeletal muscle with antibody 7H5 indicated two major sites of glycogen synthase localization. A granular localization present in the cytoplasm and a band-like staining associated with the Z-disk region of the myofibrils. Rabbit cardiac muscle presented a similar pattern though less cytoplasmic staining was apparent. An assay of subcellular fractions for glycogen synthase indicated that the enzyme in cardiac and skeletal muscles is distributed between the soluble (80-90%) and myofibrilar (10-20%) fractions of the tissues. These results provide direct evidence for the presence of glycogen synthase in subcellular fractions other than the soluble fraction of skeletal and cardiac muscles.     

Radioimmunocytochemical localization of retinal S-antigen with monoclonal antibodies

Two monoclonal antibodies (RSA1/83 and RSA2/83) were developed against a homogeneous preparation of bovine retinal S-antigen. The two hybridomas produced by mouse X mouse hybrid myeloma cells secrete immunoglobulin G. Indirect autoradiography on glutaraldehyde-fixed preparations of bovine explants was used to locate the antigenic site. Antibody RSA1/83 recognizes the antigen primarily in the apical region of the rod outer segment, while antibody RSA2/83 located the antigen both in the outer and inner segments of the rod photoreceptor cells. A distinct band of silver grains also appeared along the inner limiting membrane with both antibodies. Control explants showed no specific labeling pattern over the various retinal compartments.     

Tumor localization of Lewis lung carcinoma with radiolabeled monoclonal antibodies

Summary Mouse 6D6 IgG_2a and 5B5 IgM monoclonal antibodies which specifically bind murine lung carcinoma cells (3LL cells) were injected to healthy and tumor-bearing mice. In vivo localization was analyzed by counting the tissue radioactivity and by external gamma ray scintigraphy at various times after IV injection of ¹²⁵I- or ¹³¹I-labeled antibodies. The clearance of the two monoclonal antibodies was not modified by the presence of the tumor, and the 6D6 IgG_2a was cleared at a rate slower than the 5B5 IgM. Both antibodies gave a high specific uptake at the tumor level; the tumor-to-healthy tissue ratios were higher with the 6D6 IgG_2a than the 5B5 IgM; unspecific mouse immunoglobulins (IgG₂) did not localize in the tumor. The amount of 6D6 IgG_2a antibody still associated with the tumor after 2 days following IV injection was 10 times higher than that of 5B5 IgM, and was still high enough to localize the tumor after 5 days.Imaging experiments confirmed the ability of 6D6 IgG_2a to detect the presence of tumor cells. The targeting kinetics determined by computer analysis of camera images indicated a rapid targeting of antibodies in tumor with a maximal concentration after 4–6 h; after 48 h the background was quite low and the 6D6 IgG_2a appeared to be specifically localized in the tumor.     

Immunoelectron microscopic localization of estrogen receptor with monoclonal estrophilin antibodies

The recent production of a series of monoclonal estrophilin (estrogen receptor) antibodies recognizing estrogen receptor derived from a wide variety of animals and target tissues permits the development of immunoelectron microscopic techniques for identifying estrogen receptor. We have determined suitable conditions for the ultrastructural localization of estrogen receptor in tissue sections. Localization of receptor was observed in the euchromatin, but not in the marginated heterochromatin or nucleoli of epithelial and stromal nuclei of human endometrium. Competition studies indicate that only estrogen receptor specifically inhibits nuclear staining. The absence of any specific cytoplasmic localization at the electron-microscopic level is consistent with earlier light-microscopic observations and suggests that the majority of the cellular pool of estrophilin exists in the nucleus of hormone-responsive cells.     

Characterization of monoclonal antibodies specific for rabbit renal brush-border hydrolases: application to immunohistological localization

By use of immunodepletion studies, we characterized four monoclonal antibodies reactive with rabbit brush-border (BB) as specific for aminopeptidase N (AP), dipeptidylpeptidase IV (DPPIV), neutral endopeptidase (EP), and angiotensin-converting enzyme (ACE), and we used these antibodies for immunohistochemical detection of these four hydrolases. Expression within the kidney was studied by light and electron microscopy. All four hydrolases are expressed on the various segments of the proximal tubule. In addition, EP and DPPIV are detectable on visceral epithelial cells of the glomerulus and AP on the cells of Bowman's capsule. Outside the kidney, the four hydrolases are expressed within the digestive and genital tracts, where AP, EP, and DPPIV predominate on epithelial structures, whereas ACE is essentially located in vascular structures. The latter localization is also characteristic of ACE in the other organs studied, where clear-cut systematic distribution of the other hydrolases was often difficult to demonstrate. In addition, AP, DPPIV, and EP were detected on lymphoid cells. As compared to reports of data obtained essentially by enzymatic or immunoradiometric assays, these observations suggest considerable interspecies variations of extrarenal expression of the major BB hydrolases. This should be taken into account in attempting to define a general physiological role for a given enzyme.     

Detection of mouse ectromelia virus using the heterologous rabbit antiserum conjugated with fluorescein isothiocyanate

A technique for direct determination of cells infected with mouse ectromelia virus was elaborated using the indirect immunofluorescence method and heterologous rabbit antiserum against vaccinia virus. The proposed method can be performed in laboratories with simple equipment and does not require special virological laboratory.     

Bispecific and human disease-related anti-keratin rabbit monoclonal antibodies

Rabbit antibodies may have favorable properties compared to mouse antibodies, including high affinities and better antigen recognition. We used a biochemical and reverse immunologic approach to generate and characterize rabbit anti-phospho-keratin and anti-keratin monoclonal antibodies (MAb). Human keratins 8 and 18 (K8/K18) were used as immunogens after isolation from cells pretreated with okadaic acid or pervanadate to promote Ser/Thr or Tyr hyperphosphorylation, respectively. Selected rabbit MAb were tested by immunofluorescence staining, immunoprecipitation, and 2-dimensional gels. Keratin phospho and non-phospho-mutants were used for detailed characterization of two unique antibodies. One antibody recognizes a K8 G61-containing epitope, an important epitope given that K8 G61C is a frequent mutation in human liver diseases. This antibody binds K8 that is not phosphorylated on S73, but its binding is ablated by G61 but not S73 mutation. The second antibody is bispecific in that it simultaneously recognizes two epitopes: one phospho (K8 pS431) conformation-independent and one non-phospho conformation-dependent, with both epitopes residing in the K8 tail domain. Therefore, a reverse immunologic and biochemical approach is a viable tool for generating versatile rabbit MAb for a variety of cell biologic applications including the potential identification of physiologic phosphorylation sites.     

Characterization of mouse and human monoclonal antibodies cross-reactive with SLE serum antibodies to guanosine

Two new monoclonal antibodies, one a mouse IgM and the other a human IgM that reacted with guanosine, were compared to human serum antibodies from patients with systemic lupus erythematosus (SLE). The human monoclonal antibody was polyspecific in its binding to the nucleoside bases, whereas the mouse monoclonal antibody was relatively specific for guanosine when compared by using an enzyme-linked immunosorbent assay (ELISA). Neither antibody bound polyguanylic acid or denatured single-stranded (ss) DNA, however. Serum IgG antibodies from seven patients with SLE cross-reacted with the mouse monoclonal antibody and showed considerable specificity for guanosine. In contrast, the human serum IgG antiguanosine antibodies also bound ssDNA but not dsDNA or polyguanylic acid. Serum IgG antibodies to guanosine measured by ELISA from the seven SLE patients had a decreased response when compared to the total serum IgG response to ssDNA, and most of the antibodies that bound guanosine also bound ssDNA. These studies provide new evidence that there are specific IgG antibodies to guanosine in SLE sera that are a small fraction of the antibodies to ssDNA. Further efforts to define the role of these guanosine antibodies in SLE may provide a better understanding of the basic mechanisms responsible for the development of SLE in man.     

Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Avena shoots

While two monoclonal antibodies directed to phytochrome from etiolated oat (Avena sativa L.) shoots can precipitate up to about 30% of the photoreversible phytochrome isolated from green oat shoots, most precipitate little or none at all. These results are consistent with a report by J.G. Tokuhisa and P.H. Quail (1983, Plant Physiol. 72, Suppl., 85), according to which polyclonal rabbit antibodies directed to phytochrome from etiolated oat shoots bind only a small fraction of the phytochrome obtained from green oat shoots. The immunoprecipitation data reported here indicate that essentially all phytochrome isolated from green oat shoots is distinct from that obtained from etiolated oat shoots. The data indicate further that phytochrome from green oat shoots might itself be composed of two or more immunochemically distinct populations, each of which is distinct from phytochrome from etiolated shoots. Phytochrome isolated from light-grown, but norflurazon-bleached oat shoots is like that isolated from green oat shoots. When light-grown, green oat seedlings are kept in darkness for 48 h, however, much, if not all, of the phytochrome that reaccumulates is like that from etiolated oat shoots. Neither modification during purification from green oat shoots of phytochrome like that from etiolated oat shoots, nor non-specific interference by substances in extracts of green oat shoots, can explain the inability of antibodies to recognize phytochrome isolated from green oat shoots. Immunopurified polyclonal rabbit antibodies to phytochrome from etiolated pea (Pisum sativum L.). shoots precipitate more than 95% of the photoreversible phytochrome obtained from etiolated pea shoots, while no more than 75% of the pigment is precipitated when phytochrome is isolated from green pea shoots. These data indicate in preliminary fashion that an immunochemically unique pool of phytochrome might also be present in extracts of green pea shoots.Abbreviation ELISA enzyme-linked immunosorbent assay - mU milliunit - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome     

Structural study of GCDFP-15/gp17 in disease versus physiological conditions using a proteomic approach

Gross cystic disease fluid protein (GCDFP-15), also known as prolactin-inducible protein (PIP), is a specific breast tumor marker. GCDFP-15/PIP is also identified as gp17 and/or seminal actin-binding protein (SABP) from seminal vesicles and as extraparotid glycoprotein (EP-GP) from salivary glands. It is an aspartyl proteinase able to specifically cleave fibronectin (FN), suggesting a possible involvement in mammary tumor progression and fertilization. Other functions were attributed to this protein(s) on the basis of its ability to interact with an array of molecules such as CD4, actin, and fibrinogen. We investigated the structure of the protein(s) under disease versus physiological conditions by RP-HPLC chromatography, ProteinChip technology, and QStar MS/MS mass spectrometry. The proteins behaved differently when examined by RP-HPLC chromatography and surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) mass spectrometry, suggesting different conformations and/or tissue-specific posttranslational modifications of the proteins, although their primary structure was identical by MS/MS analysis. Both showed a single N-glycosylation site. A different N-linked glycosylation pattern was observed in pathological GCDFP-15/PIP as compared with physiological gp17/SABP protein by coupling enzymatic digestion and ProteinChip technology. Furthermore, taking advantage of ProteinChip technology, we analyzed the interaction of both proteins with CD4 and FN. We observed that the physiological form was mainly involved in the binding to CD4. Moreover, we defined the specific FN binding-domain of this protein. These data suggested that, depending on its conformational state, the protein could differently bind to its various binding molecules and change its function(s) in the microenviroments where it is expressed.     

Characterization of mouse monoclonal antibodies to human interferon-gamma

Mouse monoclonal antibodies (mAb) to human interferon-gamma (HuIFN-gamma) were characterized. The mAbs studied--E4-18, G4-15, and SAT-1--which are all IgGl-type, reacted to all HuIFN-gamma molecular species, both glycosylated and non-glycosylated. Affinity constants calculated of E4-18 and G4-15 didn't have considerable differences for both kinds of HuIFN-gamma (1-3 x 10(8) liter/mol), but SAT-1 had a difference--a higher value (10(10) liter/mol) for the former than for the latter (8 x 10(8) liter/mol). In epitope specificity, the results suggested that E4-18 and G4-15 recognized an overlapped region remote from the region of SAT-1. Competition experiment using synthetic peptides suggested that epitope of G4-15 is around N9-26 of the HuIFN-gamma sequence. Those mAbs could be used for sandwich radioimmunoassay of HuIFN-gamma using double mAbs in two combinations, one (G4-15/E4-18) based on dimer forms of HuIFN-gamma and the other (SAT-1/E4-18) based on epitope difference. The mAbs are all neutralizing antibodies in which SAT-1 neutralized at a lower concentration than did G4-15, and at a much lower one than did E4-18. The receptor binding of HuIFN-gamma was inhibited by mAbs G4-15 and SAT-1. Efficacy of G4-15 and SAT-1 for the inhibition correspond with that for neutralization.     

Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies.

Two hybridomas (H3 and D3) secreting monoclonal neutralizing antibody to intact poliovirus type 1 (Mahoney strain) were established. Each antibody bound to a site qualitatively different from that to which the other antibody bound. The H3 site was located on intact virions and, to a lesser extent, on 80S naturally occurring empty capsids and 14S precursor subunits. The D3 site was found only on virions and empty capsids. Neither site was expressed on 80S heat-treated virions. The antibodies did not react with free denatured or undenatured viral structural proteins. Viral variants which were no longer capable of being neutralized by either one or the other antibody were obtained. Such variants arose during normal cell culture passage of wild-type virus and were present in the progeny viral population on the order of 10(-4) variant per wild-type virus PFU. Toluene-2,4-diisocyanate, a heterobifunctional covalent cross-linking reagent, was used to irreversibly bind the F(ab) fragments of the two antibodies to their respective binding sites. In this way, VP1 was identified as the structural protein containing both sites.