Monoclonal antibody to thyroid transcription factor-1: production, characterization, and usefulness in tumor diagnosis.

Thyroid transcription factor-1 (TTF-1), a member of the NKx2 family of homeodomain transcription factors, is expressed in epithelial cells of the thyroid gland and the lung. To produce monoclonal antibodies specific for TTF-1, the polypeptide was expressed in E. coli and purified utilizing affinity chromatography of a polyhistidine-tagged TTF-1 fusion protein. Splenocytes from BALB/c mice immunized with recombinant TTF-1 were fused with P3x/63Ag8.653 myeloma cells to produce hybridomas. Tissue culture supernatant was screened for anti TTF-1 activity by ELISA employing recombinant TTF-1 as antigen. Hybridomas producing high-affinity antibodies were subcloned by limiting dilution. Antibodies from tissue culture fluid from an IgG1 clone (8G7G3/1) that stained the nuclei of paraffin-embedded human thyroid tissues were precipitation-purified and further characterized. The antibody stained a single 40-kDa polypeptide in immunoblots of nuclear extracts or lysates of cell lines known to express TTF-1 mRNA. MAb 8G7G3/1 also stained nuclei of tissue in a highly specific manner consistent with the pattern of expression obtained with an established polyclonal TTF-1 antibody and by in situ hybridization. MAb 8G7G3/1 was used for TTF-1 immunohistochemistry of human adenocarcinomas of the lung, colon, and breast as well as small cell carcinomas of the lung. TTF-1 was detected in primary lung adenocarcinomas and small cell carcinomas and was absent in colon and breast carcinomas. These findings demonstrate that anti-TTF-1 MAb 8G7G3/1 specifically binds TTF-1 in cell extracts and tissues and can be used to distinguish between lung and nonlung origin of a tumor.

Human surfactant protein-A contains blood group A antigenic determinants.

A major blood group antigenic epitope was identified on human pulmonary surfactant protein A (SP-A). MAb and polyclonal antibodies generated against purified human SP-A aggregated blood group A human erythrocytes and immunostained epithelial cells in a variety of human tissues, consistent with the tissue distribution of major blood group antigens. SP-A MAb (MAb-8) agglutinated red cells and immunostained tissues from A or AB blood groups, but did not react with cells or tissues from O or B individuals. MAb-8 immunostaining of tissue from blood group A individuals was ablated by incubation with blood group A red cells. MAb and polyclonal antibodies directed against A blood group antigens reacted strongly with purified SP-A obtained from a blood group A individual with alveolar proteinosis. MAb and polyclonal antibodies specific for B blood group antigen failed to react with SP-A from this patient or from patients who were in blood group B. Reactivity of anti-blood group MAb was lost after treatment of SP-A with endoglycosidase-F, demonstrating its reactivity with an epitope dependent on the asparagine-linked oligosaccharide at asparagine 187. Reactivity of MAb-8 with SP-A persisted after endoglycosidase-F treatment, but was lost after digestion with collagenase as assessed by Western blot after SDS-PAGE. Reactivity of MAb to SP-A was sensitive to beta-elimination, supporting the presence of another blood group antigenic site distinct from the epitope dependent on the asparagine-linked carbohydrate. The finding that the SP-A molecule contains a major blood group epitope has implication for the clinical use of surfactant replacement preparations and diagnostic reagents based on this protein.

Comparison of properties of monoclonal and polyclonal antibodies against the light chain of human high molecular weight kininogen.

A murine hybridoma monoclonal line-secreted antibody (C3G5) against the light chain of human high molecular weight kininogen (HMWK), which consisted of gamma-1 kappa isotype, was largely composed of 190 kd molecules, and gave an optimal reaction when used in an equimolar concentration with HMWK. It did not influence the initial digestion of HMWK or the amount of kinin released by kallikrein. Pretreatment of HMWK with C3G5 antibodies did not augment or inhibit its coagulant properties, nor did pretreatment interfere with its adsorption on kaolin or on the ion-exchange resin diethylaminoethyl (DEAE) Sephadex A-50. The epitope that reacted with this antibody was localized to a portion of the light chain within 6 kd of the carboxy-terminus of the molecule near the single cysteine of this chain, at residue 614. This monoclonal antibody reacted with an epitope that was stable during early plasmin digestion. The amounts of antigens reactive with a polyclonal antibody to the light chain were increased during the same period of plasmin digestion. Prolonged plasmin digestion reduced the amounts of both types of light chain antigens. During kallikrein digestion, the amount of epitope reactive with C3G5 antibody was unaffected, whereas the concentration of antigens detected with polyclonal anti-light chain antibodies was increased throughout digestion. Therefore, light chain antigens reactive with the polyclonal antibody are probably in the interior of the uncleaved molecule and become exposed after initial cleavage, which probably facilitates changes in the conformation of the molecule. The epitope reactive with monoclonal antibodies is probably on the surface of the uncleaved molecule.

Surfactant-associated protein inhibits phospholipid secretion from type II cells.

Secretion of [3H]phosphatidylcholine ([3H]PC) from isolated rat pulmonary type II epithelial cells was inhibited by the surfactant-associated protein of Mr = 35,000 (SAP-35) purified from canine lung surfactant. SAP-35 inhibited [3H]PC secretion in a dose-dependent manner and significantly inhibited basal, phorbol ester, beta-adrenergic, and P2-purinergic agonist-induced [3H]PC secretion. SAP-35 significantly inhibited [3H]PC secretion from 1 to 3 h after treatment. The IC50 for inhibition of [3H]PC secretion by canine SAP-35 was 1-5 X 10(-6) g/ml and was similar for inhibition of both basal and secretagogue-stimulated release. Heat denaturation of SAP-35, addition of monoclonal anti-SAP-35 antibody, reduction and alkylation of SAP-35, or association of SAP-35 with phospholipid vesicles reversed the inhibitory effect on secretagogue-induced secretion. Inhibitory effects of SAP-35 were observed 3 h after cells were washed with buffer that did not contain SAP-35. Although SAP-35 enhanced reassociation of surfactant phospholipid with isolated type II cells, its inhibitory effect on secretion of [3H]PC did not result from stimulation of reuptake of secreted [3H]PC by type II cells. The inhibition of phospholipid secretion by SAP-35 was also not due to inhibition of PC or disaturated PC synthesis by SAP-35. SAP-35, the major phospholipid-associated protein in pulmonary surfactant, is a potent inhibitor of surfactant secretion from type II cells in vitro and may play an important role in homeostasis of surfactant in the alveolar space.

beta 2-microglobulin in human tumors heterografted into athymic mice.

Beta 2-microglobulin (beta 2m) content of several human tumor lines implanted into athymic mice was investigated. The concentration of this protein was found to vary greatly from tumor to tumor. The growth rate of the tumors was inversely related to the amount of the extractable beta 2m. The fastest growing, undifferentiated tumors contained the least amount of beta 2m.

Release of beta 2-microglobulin by human tumors grown in nude mice.

Human tumors implanted subcutaneously into athymic mice produced human beta 2-microglobulin which was readily identified and quantified in mouse plasma. Implanted solid tumors of the lines Clouser, SW480, Hep-2, Capan-1, and HT-29 released amounts of beta 2-microglobulin directly related to tumor mass. The extractable beta 2-microglobulin per gram of tumor was constant for each cell line, but there was a 100-fold variation between the lines. In general, the plasma level of beta 2-microglobulin correlated with the amount of free beta 2-microglobulin extracted from each tumor. From these observations it can be concluded that the variablility observed in the circulating levels of beta 2-microglobulin among cancer patients is related to the beta 2-microglobulin content of the tumor and the total tumor mass.

Distribution of the phosphoenolpyruvate: glucose phosphotransferase system in bacteria.

A survey of the occurrence of the phosphoenolpyruvate-dependent glucose phosphotransferase system was carried out in a number of bacteria, representing both gram-positive and gram-negative facultative anaerobic and strictly aerobic types. The system was found to be present in representatives of genera that are characteristically facultative anaerobes, but the system was absent in members of those genera that are strictly aerobic. Thus, although the phosphoenolpyruvate phosphotransferase system is an important system for the transport of sugars in bacteria carrying out anaerobic glycolysis, it plays no role in sugar transport by those organisms having a strictly oxidative physiology. A fundamentally different system, probably not involving phosphorylation during transport, is indicated in this latter group.