Immunohistochemical localization of rat submandibular gland esterase B (homologous to the RSKG-7 kallikrein gene) in relation to other serine proteases of the kallikrein family.

The rat submandibular gland contains several members of the kallikrein family. In the present study we purified and raised an antiserum against one of these enzymes, i.e., esterase B, which was first described by Khullar et al. in 1986. N-terminal amino acid analysis revealed complete homology between esterase B and the kallikrein family gene RSKG-7. For characterization of the antiserum, flat-bed isoelectrofocusing with immunoblotting was superior to immunoelectrophoresis and double immunodiffusion in detecting and identifying crossreacting proteins. This was due to the fact that kallikrein-like enzymes were readily separated by isoelectrofocusing, and immunoreactivity was easily detected by the sensitive peroxidase-anti-peroxidase staining after blotting onto nitrocellulose membrane. Immunohistochemical controls were carried out accordingly, including homologous as well as crossreacting antigens. In the submandibular gland, esterase B was detected exclusively in all granular convoluted tubular cells, co-localized with tissue kallikrein and tonin. Some staining was also observed in striated duct cells; however, this staining reaction was induced by cross-reactivity with kallikrein, since staining was abolished by addition of kallikrein as well as esterase B to the primary antiserum. It was therefore concluded that like tonin and antigen gamma, but unlike kallikrein, esterase B was not detected in the striated ducts of the submandibular, parotid, or sublingual glands. This separation in anatomic distribution between esterase B and kallikrein may indicate that prokallikrein activation is not the only biological function of esterase B.     

Antigenic analysis of Chlamydiae by two-dimensional immunoelectrophoresis. II. A trachoma-LGV-specific antigen.

Two-dimensional immunoelectrophoresis was utilized to study precipitins in hyperimmune rabbit serum made against chlamydiae and from patients with chlamydial infections. An antigen of Triton X-100-solubilized L2/434/Bu organisms with an electrophoretic mobility of 0.65 relative to bovine serum albumin at pH 8.6 was excised from the agarose gel of electrophorograms as antigen-antibody complexes and used to immunize rabbits. A monospecific antiserum to antigen 0.65 was obtained that reacted with Trachoma-LGV strains L2/434/Bu, B/TW-5/OT, and K/UW-31/Cx, but not with the mouse pneumonitis (Nigg) strain or the psittacosis strain meningopneumonitis (Cal-10). The Trachoma-LGV specificity of antigen 0.65 was further shown by indirect immunofluorescence straining with the monospecific antiserum of chlamydial inclusions in infected HeLa cells. Precipitins with a specificity for antigen 0.65 were indentified in 15 of 18 sera from patients with diagnosed Chlamydia trachomatis infections LGV, trachoma, nongonococcal urethritis, and nongonococcal cervicitis by using monospecific antiserum to antigen 0.65 in the peak suppression test. Thus, antigen 0.65 appears to be a Trachoma-LGV-specific antigen that has considerable promise for serodiagnosis.     

Further observations on the specificity of antigen 5 of Echinococcus granulosus.

The presence of IgE antibodies to antigen 5 of Echinococcus granulosus was detected by means of radioimmunoelectrophoresis in the sera of two of six patients infected with E. multilocularis. Sera from three of these patients gave a precipitin band in gel diffusion tests identical to that produced by a monospecific rabbit anti-E. granulosus antigen 5 serum, when tested against whole hydatid fluid. Sera from 19 individuals infected with Fasciola hepatica, 20 with Schistosoma mansoni, and 5 with with Taenia saginata showed no detectable antibodies against antigen 5 of E. granulosus, The monospecific rabbit anti-E. granulosus antigen 5 serum did not react in immunodiffusion with homologous antigen when absorbed with either 4 mg/ml of whole hydatid fluid or with 200 mg/ml of a soluble E. multilocularis extract. Absorption of the monospecific antiserum with crude antigens of either F. hepatica, Onchocerca volvulus, S. mansoni, or T. saginata did not abolish the reaction with antigen 5. It appears, therefore, that antigen 5 can no longer be considered specific for E. granulosus, but is also present in E. multilocularis. In the light of this observation, some reevaluation of immunodiagnostic tests in hydatid disease will be necessary.     

Immunohistochemical detection of the sex steroid-binding plasma protein in human mammary carcinoma cells

A sex steroid-binding plasma protein-like antigen has been detected in human mammary carcinoma cells. A monospecific antiserum was used, and this protein was located mainly on the cytoplasmic membranes. These results are in agreement with a recent hypothesis according to which steroid hormones could be carried into cells by specific binding plasma proteins.     

Phosphatase active antigens in sea urchin eggs and embryos. II. A comparison between the activities in unfertilized eggs and plutei.

Phosphatase activities in sea urchin eggs and plutei were investigated by means of histochemical staining of immunoprecipitates. Two protein fractions were obtained by extraction in a hypotonic medium and by detergent treatment of the residual pellet. Three distinctly different phosphatase activities were discerned, nucleoside diphosphatase (EC 3.6.1.6.), acid phosphatase (EC 3.1.3.2.) and alkaline phosphatase (EC 3.1.3.1.). The nucleoside diphosphatase activity, which was confined to one antigen, was present in both water soluble and detergent extracts and at roughly the same concentration in eggs and plutei. By means of a monospecific antiserum the immunological identify of this antigen was established in all instances. The acid phosphatase activity, which was displayed by ten detergent extracted antigens in eggs, was only found in five detergent extracted antigens in plutei. This decrease in number of enzyme active antigens was also reflected by a general decrease in number of enzyme active antigens was also reflected by a general decrease in activity as assessed by quantitative determinations. Furthermore, by means of absorbed antisera it was established that two or three of the acid phosphatase active antigens were "egg specific". Another acid phosphatase active antigen, which was common to both developmental stages, was investigated by a monospecific antiserum. While this antigen was found in both soluble fractions, it was only enzymatically active when extracted with detergent. Alkaline phosphatase active antigens were only found in the detergent extract of plutei. However, immunoprecipitates with this activity appeared both with antiserum against unfertilized eggs and with antiserum against plutei. This suggests that the egg contained the antigens in an enzymatically inactive form.     

Immunological studies of canine ovarian antigens

Studies were conducted to determine the feasibility of producing canine ovary antiserum in the rabbit and to characterize the antigenic composition of the canine ovary. Ovaries from dogs were obtained and corpora lutea (CL) macroscopically removed. Following homogenization of ovaries, adult male rabbits were immunized by injecting the ovarian preparation. Unabsorbed antiserum cross-reacted with canine ovarian extract and with other reproductive and non-reproductive organ extracts to form precipitin bands. Species cross-reactivity was also observed by testing the unabsorbed antiserum with extracts from organs of the cat and rat. Absorption of antiserum with canine liver and lung extracts removed antibodies not specific to the canine ovary. One band was observed when such absorbed antiserum was allowed to react with the canine ovarian extract. The absorbed antiserum produced no bands with extracts from other canine reproductive and non-reproductive organs tested. These experiments suggested that the canine ovary contained at least 1 organ-specific antigen. This organ-specific antigen was located in the isolated canine ovarian cells by the immunofluorescence technique.     

Corticosteroid side-chain isomerase in mouse organs: kinetic and immunologic studies

We have investigated the distribution of corticosteroid side-chain (CSC) isomerase in the tissues of mice using as criteria its enzyme activity and immunoreactivity with monospecific polyclonal antibodies generated in rabbits. CSC isomerase was present in all organs examined. The liver and kidney contained the highest activity. The strain-dependent differences that we had previously reported for liver (i.e., BALB/c greater than C57BL/6) extended to the other organs, including the kidney, brain, heart, muscle, pancreas, testis, thymus, and lung. Western blot analysis showed a single antigen, identical in all tissues, corresponding in mobility to purified CSC isomerase. The intensities of the bands were generally proportional to enzyme activities. Titration of homogeneous enzyme with the IgG fraction of antiserum (unfractionated serum had some CSC isomerase activity) caused an increase in activity, followed by rapid inactivation after the addition of more antiserum. The broad distribution of CSC isomerase suggests that the ketol-aldol interconversion of the CSC may play a role other than, or in addition to, initiating metabolic inactivation of corticosteroids.     

Preparation of monospecific antisera to IgA in laboratory animals using cascade immunization without preliminary isolation of the antigen

The proposed method of "cascade" immunization for the preparation of monospecific antisera to IgA of the experimental animals (mice, rats, guinea-pigs) requires no isolation and purification of an antigen, since it is based on using precipitation lines containing IgA. The subsequent steps of the method are the following: 1) preparation of a polyspecific antiserum against one of the secretions or IgA-containing serum fraction; 2) preparation of precipitation lines using this antiserum and some other secretions; 3) immunization of other rabbits with these precipitation lines to obtain monospecific anti-IgA antiserum. The antisera obtained do not require any additional absorption except for the removal of anti-IgA antibodies. The method can be used for the preparation of monospecific antisera to any other serum or secretion proteins.     

Arylamidases of rat liver and chemically induced hepatomas. II. Preparation and characterization of monospecific antisera against two distinct arylamidase-active antigens.

Monospecific antisera were prepared against the most prominent arylamidase (alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2) active antigen in plasma membranes (the plasma membrane arylamidase) and lysomal content (the lysosomal content arylamidase), respectively. Plasma membrane extract and lysosomal content were allowed to react in crossed immunoelectrophoresis against their homologous antisera. The electrophoretic plates were washed extensively, dried and subsequently stained for arylamidase activity.The particular immunoprecipitates were thus identified and could be excised to be used for immunizations. The two resulting antisera precipitated the arylamidase used for immunization, but failed to be monospecific as they precipitated additional antigens. These antisera with restricted specificity against some plasma membrane and lysosomal content antigens, respectively, were used to produce immunoprecipitates intended for new attempts to prepare monospecific antisera by a second cycle of immunizations. A monospecific antiserum against the plasma membrane arylamidase was thus obtained, while a third cycle of immunizations was needed to get a monospecific anti-lysosomal content antiserum. The plasma membrane arylamidase showed ATPase activity also after precipitation with the monospecific antiserum, thus still retaining its characteristics as a multienzyme complex.     

Immunoelectrophoresis of mycobacterial antigens

Polyvalent antiserum to culture filtrate of H37 Ra M. tuberculosis was raised in rabbits. Monospecific antiserum was raised against M. tuberculosis antigen-5, prepared from the culture filtrates by immunoabsorbent affinity chromatography. On immunoelectrophoresis, antigen-5 demonstrated single precipitin arc against polyvalent and monospecific antisera. The culture filtrate antigen demonstrated multiple precipitin arcs against polyvalent antiserum and single precipitin are against monospecific antiserum. Antigen-5 could be isolated and characterized from the culture filtrate of H37 Ra M. tuberculosis. Immunoelectrophoresis could be one of the method to characterize the mycobacterial antigens prepared in the laboratory.     

Specific antigen of granular cells of the human endometrium

Rabbits were immunized by homogenates of endometrium obtained from women during 10-12 weeks of gestation. A specific antiserum was obtained by absorption of the crude antiserum by blood cells and plasma proteins of men with different kinds of ABO and Rh antigens, till disappearance of positive reaction with men's serum protein in the Ouchterlony test. Such an adsorbed specific antiserum continued to react with the sera of pregnant women. Two antigens, numbers 1 and 2, respectively, were determined by the Ouchterlony test. Another group of rabbits was immunized by antigens detected in the precipitation test. A monospecific antidecidual antiserum (ADS 1092) was obtained against number 2 antigens. This antiserum revealed only one antigen in sera of women with gestation and did not react with sera of non-pregnant women. In the slides of endometrium of pregnant women of 10-12 weeks of gestation ADS 1092 had a strong positive reactive with the cytoplasm of one type of endometrium cells. The immunomorphological analysis by the non-direct Coons test and the PAP-test permits to identify cells with the positive reaction as granular cells. It is concluded that the granular cells may be a source of one of the serum antigens detected in women with gestation.     

Identification of an 80-kilodalton membrane glycoprotein important for human T-cell leukemia virus type I and type II syncytium formation and infection.

Human T-cell leukemia virus type I and type II (HTLV-I and HTLV-II, respectively) infect certain sublines of the BJAB human B-cell line. We observed that the WH subline, but not the CC/84 subline, of BJAB cells were infectible by cell-free HTLV-I or HTLV-II and formed syncytia with cells infected by these retroviruses. This suggests that the BJAB-CC/84 cells possibly lack a membrane molecule(s) important for syncytium formation and infectibility. In order to identify this antigen, we generated polyclonal anti-BJAB-WH antisera which were adsorbed on BJAB-CC/84 cells. The adsorbed antisera bound only BJAB-WH and BJAB-CC/79 cells as demonstrated by complement-dependent cytotoxicity and flow cytometric assays. Furthermore, this adsorbed antisera bound several human T-cell clones, including SupT-1, as determined by flow cytometric assays. The adsorbed antiserum was monospecific as it immunoprecipitated only one 78- to 80-kDa protein from lysates of metabolically labeled BJAB-WH, BJAB-CC/79, and SupT-1, but not BJAB-CC/84, cells. The monospecific antisera detected a glycoprotein composed of a 64- to 66-kDa core protein containing tunicamycin-sensitive N-linked oligosaccharides. This membrane glycoprotein appears to be involved in HTLV-I- and HTLV-II-induced fusion and infection, as the monospecific antisera were capable of inhibiting both of these processes. The monospecific antisera diluted 1:50 and 1:90 inhibited 85 to 90% of syncytium formation induced in BJAB-WH, BJAB-CC/79, and SupT-1 cells cultured with HTLV-I- or HTLV-II-infected MT2, MoT, or FLW human T- or B-cell lines. At the same dilution, antisera inhibited 70 to 80% of infection of BJAB-WH cells by cell-free HTLV-I or HTLV-II. Thus, these studies indicate a role for a 78- to 80-kDa glycoprotein in HTLV-I or HTLV-II infection and syncytium formation.     

Role of a carbohydrate component in the formation of immunochemical determinants of carcinoembryonic antigen

Carcinoembryonic antigen (CEA) isolated from metastases of colon adenocarcinoma was subjected to deglycosylation with liquid hydrogen fluoride. The protein fraction obtained (PF CEA) was used for to prepare monospecific antiserum to PF CEA. Comparative studies of CEA, PF CEA, and non-specific cross-reacting antigen (NCA-1) have been carried out using monospecific antisera. Circular dichroism spectra of CEA and PF CEA have been studied. The data obtained suggest that some immunodominant regions of CEA are topographic, and their formation needs a specific conformation of the macromolecule, which is stabilized by the sugar moiety.     

Trypanosoma brucei: a membrane-associated protein in coated endocytotic vesicles

Membrane proteins were isolated from purified Trypanosoma brucei coated endocytotic vesicles by phase separation with Triton X-114. The largest abundant membrane protein was a doublet band with a molecular mass of about 77 kDa. A specific antiserum was prepared against this protein by immunization with antigen bands excised from sodium dodecyl sulfate-polyacrylamide gels. Immunoblot analyses with this antiserum showed that the 77-kDa protein was present in other T. brucei, in T. congolense, and in T. vivax bloodstream-stage parasites but absent from procyclic (tsetse fly midgut)-stage trypanosomes. Antigenically related molecules of 58, 300, and 15.5 kDa were also detected. The 300- and 15.5-kDa molecules were not in purified coated vesicles; they were detected in whole bloodstream- and procyclic-form T. brucei organisms. Immunofluorescent studies localized the antigen to the region between the flagellar pocket and the nucleus of bloodstream-form parasites. Ultrastructurally, the antigen was detected on membranes of endosomes and lysosome-like structures that contained endocytosed markers.     

Sheep haemopexin: immunological cross-reactivity with other vertebrate sera and evidence for the absence of haemopexin in some mouflon

A monospecific antiserum to sheep haemopexin was produced in rabbits. By using this antiserum, as well as absorbed antisera to sheep and mouflon serum proteins, it was proved that some mouflon sera lack haemopexin. In immunodiffusion, when using the monospecific antiserum, immunoprecipitates were present only in species' belonging to suborder Ruminantia. None of the other mammalian, nor any of the avian, reptilian, amphibian and fish species tested, showed reaction.     

Pancreatic cholesterol esterases. 1. Pancreatic cholesterol esterase induction during maturation

The activities of pancreatic cholesterol esterase from calf and cow pancreas were examined in detail. A 1300-fold enhancement of enzymatic activity was found after maturation, even though cholesterol esterase activity levels in other organs did not change from the juvenile to the adult species. Radioimmunoassays also showed that the calf pancreas contained at least 100-fold less cholesterol esterase protein. Decreased amounts of protein were not due to enhanced proteolysis, since cytosol from cow pancreas degrades exogenously added cholesterol esterase faster than that from calf pancreas. Rather, enhancement of pancreatic cholesterol esterase activity associated with bovine maturation was the result of specific, increased synthesis of a 72-kDa enzyme. This labile 72-kDa cholesterol esterase species was purified to homogeneity by a two-step process in 75% yield and is the major form of bovine pancreatic cholesterol esterase (99%). A much less abundant 67-kDa species, accounting for less than 1% of total pancreatic cholesterol esterase activity, was also purified to homogeneity in a similar two-step process. These results demonstrate that a specific form of pancreatic cholesterol esterase is induced during maturation, and they bear importantly on understanding juvenile cholesterol metabolism as related to dietary absorption of this sterol.     

Tissue distribution of a novel neurotensin-degrading metallopeptidase. An immunological approach using monospecific polyclonal antibodies.

A monospecific polyclonal antiserum was raised against a recently purified rat brain neurotensin-degrading metallopeptidase. The purified IgG fraction immunoprecipitated the peptidase and inhibited its proteolytic activity. Western blot analyses revealed that the immune fraction recognizes only one protein in rat brain homogenates, and this corresponds closely to the purified enzyme. The IgG displayed a restricted specificity towards the peptidase from murine origin. In the rat, the neurotensin-degrading enzyme was widely distributed throughout peripheral organs with the noticeable exception of the duodenum. In addition, the peptidase was detected in various cell lines or membrane preparations of neural or extraneural origin in which it had been previously characterized by means of biochemical methods. In light of this widespread distribution, the putative role of the peptidase in the metabolism of neuropeptides is discussed.     

Erythrocyte membrane antigen expression during friend cell differentiation: Analysis of two non-inducible variants

Erythrocyte membrane antigens have been detected on induced Friend erythroleukemic cells with a rabbit antiserum raised against mouse erythrocyte membranes. The antibody specificities of this antiserum have been quantitatively analyzed using a cellular radioimmunoassay. After absorption with thymocytes, the rabbit anti-erythrocyte membrane serum bound to dimethylsulfoxide (DMSO)-induced Friend erythroleukemic cells and to mouse erythrocytes but not to uninduced Friend cells or thymocytes. Reciprocal inhibition studies demonstrated that, following complete thymocyte absorption, the antiserum detected similar antigenic specificities, termed erythrocyte membrane antigens (EMA), on both mature erythrocytes and induced Friend cells. The expression of these erythrocyte membrane antigens was also induced on Friend cells by other agents, such as ouabain and dimethylacetamide (DMA). In contrast, exogenous hematin, which did not induce hemoglobin synthesis in the Friend cell clones used in this study, also did not induce erythrocyte membrane antigen expression. Two independently derived variant clones which do not produce hemoglobin in reponse to DMSO were analyzed for their ability to produce erythrocyte membrane antigens in response to various inducers of Friend cell differentiation. Clone TG-13 is not inducible by DMSO or hematin but is weakly induced by DMA for both hemoglobin production and erythrocyte membrane antigen expression. Another variant clone, M18, was also analyzed. This clone does not synthesize detectable hemoglobin when grown in either DMSO or hematin alone, but undergoes extensive hemoglobin synthesis when grown in medium containing both DMSO and hematin. M18 does, however, express erythrocyte membrane antigens when grown in DMSO alone: the presence of hematin and DMSO together in the growth medium does not enhance expression of these antigens. Thus M18 appears to be defective for hemoglobin inducibility, and this defect can be overcome by exogenous hematin; however, the expression of erythrocyte membrane antigens is not affected by this block in hemoglobin synthesis. The results with the variant clones are discussed in terms of a program for Friend cell differentiation in which the induction of hemoglobin synthesis and erythrocyte membrane antigen expression are under both co-ordinate and separate controls.     

猴ESc—615基因重组蛋白质片段的表达纯化和多克隆抗体的制备

ESc 6 15是从猕猴中克隆到的一个在附睾中特异性表达的新基因。为了从蛋白质水平深入研究其在精子成熟中的作用 ,在大肠杆菌中表达了该蛋白质的一条含 310个氨基酸的肽段 ,并用其免疫新西兰大白兔 ,得到了滴度为 10 0 0 0 0的抗血清 ;用Western印迹方法鉴定发现该抗血清可检测到 3ng的抗原量 ;并在大鼠附睾组织抽提液中检测到一种能与该抗血清作用的大小约 6 3kD的蛋白质 ,此蛋白质大小与作者实验室在大鼠中克隆到的此基因的同源蛋白质相同 ;利用该抗体通过免疫组化分析确定了ESc 6 15为分泌蛋白质 ,并能与精子结合 ;该抗血清经抗原吸附后阳性结果消失。该高滴度多克隆抗体的获得为ESc 6 15功能的探索提供了一条途径。     

Stimulation of ornithine decarboxylase synthesis in the rat thyroid

High titer antiserum to hepatic ornithine decarboxylase was prepared by employing enzyme·monospecific antibody complex as the immunizing antigen. This new antiserum preparation was successfully labeled with ¹²⁵I and was found to retain its specific immune properties. Iodinated antiserum was used to precipitate thyroid ornithine decarboxylase induced by a mixture of thyroid stimulating hormone and methyl xanthine in rat thyroids $\text{in vitro}$. ¹²⁵I-labeled antibody incorporation into the enzyme antibody complex after induction $\text{in vitro}$ showed an increase which paralleled the increase in enzymatic activity and thus suggested $\text{de novo}$ synthesis of thyroid enzyme protein.