Characterization of a plasma factor having opiate and immunoactivity like beta-endorphin.

A factor from human plasma having opiate-like activity was characterized in the present study. In addition to its ability to inhibit the binding of [³H]-methionine-enkephalin to opiate receptors, it also cross-reacted with two β-endorphin specific anti-sera. Compared with β-endorphin, the plasma factor had a shorter action on inhibiting the contraction of the guinea pig ileum. By gel-filtration chromatography, the size of this factor was intermediate between that of β-endorphin and methionine-enkephalin.     

A new approach for quantitative evaluation of cross-reactivity of steroids with an antiserum by radioimmunoassay: application to a highly specific antiestriol

The study of several antiestriol antisera in the presence of a series of analogues of estriol has led to a critical discussion of the classical cross-reaction test. A more accurate test (CR₁ ng) which permits a more precise determination of specificity of antisera is described.     

Characterization of an antiserum against the glucocorticoid receptor

An immunoglobulin (IgG) fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor from rat liver cytosol contained specific antibodies to glucocorticoid receptor. This was shown following incubation of the [³H]triamcinolone acetonide-glucocorticoid receptor (TA-GR) complex with the IgG fraction by (I) adsorption of the [³H]TA-GR-antibody complex to protein A linked to Sepharose, (II) an increased sedimentation rate of the [³H]TA-GR-antibody complex compared to that of the [³H]TA-GR complex, and (III) an increased molecular size of the [³H]TA-GR-antibody complex when compared to that of the [³H]TA-GR complex as judged from gel filtration. The antibody fraction was characterized with regard to titer, cross-reactivity and specificity. The antibodies cross-reacted with the glucocorticoid receptor from various rat tissues (liver, thymus and hippocampus), as well as with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus. In the rat liver, the antibody bound to both the nuclear and the cytosolic glucocorticoid receptor (Stokes radius 6.1 nm). It did not cross-react with the proteolytic fragments of the glucocorticoid receptor, the 3.6 nm complex or the 1.9 nm complex. Binding of the antibodies was not seen to the androgen, estrogen or progestin receptors in rat to rat serum transcortin. With an indirect competitive ELISA (enzyme-linked immunosorbent assay) combined with various separation techniques, based on different physiocochemical principles, it was shown that the glucocorticoid receptor was the only detectable antibody binding protein from rat liver cytosol using this assay system. These findings also indicate an immunochemical similarity between glucocorticoid receptors in different tissues as well as in different species, but not between glucocorticoid receptors and other steroid hormone receptor proteins. The cytosolic and nuclear glucocorticoid receptors in rat liver were shown to be immunochemically similar.     

Development of a radioimmunoassay for prostaglandin D2 using an antiserum against 11-methoxime prostaglandin D2

A sensitive and specific radioimmunoassay for prostaglandin D2 has been developed using its stabilized 11-methoxime derivative, which was obtained after treatment of prostaglandin D2 with methoxamine-HCl. The antiserum was obtained after injection of prostaglandin D2-methoxamine coupled to bovine serum albumin. A (125I)-Histamide prostaglandin D2-methoxamine tracer was prepared by iodination of the corresponding histamide, followed by thin layer chromatography purification. The sensitivity of the assay was 280 femtomoles per ml at 50% displacement. The cross reactivities were 15% with prostaglandin D1-methoxamine and less than 0.20% with other prostaglandins. Determination of the half-life of prostaglandin D2 in a solution containing albumin was also carried out, since it has been shown to catalyze prostaglandin D2 destruction. The unstability of this prostaglandin is due to the presence of a beta-hydroxy ketone group, and all prostaglandins possessing this labile moiety could be stabilized by such a derivatization before developing a radioimmunoassay.     

Characterization of a xenogeneic antiserum raised against the fetal germ cells of the mouse: cross-reactivity with embryonal carcinoma cells.

A xenogeneic antiserum (PG-1) raised against the germ cells of the 13.5d p.c. mouse conceptus reacts with the fetal germ cells of both sexes and adult male germ cells, but not with any nongerminal tissue that has been tested. PG-1 can also react with the EC cells of four different teratocarcinoma cell lines. There are, however, marked differences in the absorptive capacity of the different EC cell lines. The antigen(s) recognized by the antiserum are confined to only a proportion of cells in some EC lines. This antiserum has a number of potential applications in studies of the origin and development of the mouse germ cell lineage.     

Development of a radioimmunoassay for prostaglandin D2 using an antiserum against 11-methoxime prostaglandin D2

A sensitive and specific radioimmunoassay for prostaglandin D₂ has been developed using its stabilized 11-methoxime derivative, which was obtained after treatment of prostaglandin D₂ with methoxamine-HCl. The antiserum was obtained after injection of prostaglandin D₂-methoxamine coupled to bovine serum albumin. A (¹²⁵I)-Histamide prostaglandin D₂-methoxamine tracer was prepared by iodination of the corresponding histamide, followed by thin layer chromatography purification. The sensitivity of the assay was 280 femtomoles per ml at 50% displacement. The cross reactivities were 15% with prostaglandin D₁-methoxamine and less than 0.20% with other prostaglandins. Determination of the half-life of prostaglandin D₂ in a solution containing albumin was also carried out, since it has been shown to catalyze prostaglandin D₂ destruction. The unstability of this prostaglandin is due to the presence of a β-hydroxy ketone group, and all prostaglandins possessing this labile moiety could be stabilized by such a derivatization before developing a radioimmunoassay.     

Radioimmunoassay of pancreatic polypeptide in mammalian and submammalian vertebrates using a carboxyl-terminal hexapeptide antiserum

Pancreatic polypeptide (PP) immunoreactivity in acid-ethanol extracts of the pancreas of representative species of mammals, birds, reptiles, amphibians, and fish was studied by a radioimmunoassay (RIA) that utilizes an antiserum which cross-reacts exclusively with the COOH-terminal hexapeptide of PP (CTPP). PP immunoreactivity in acid-ethanol extracts of rat nonpancreas tissues (stomach, duodenum, skeletal muscle, brain) was also examined. Significant concentrations of PP immunoreactivity were detected in the pancreatic extracts of all species, except fish. Appreciable quantities of PP immunoreactivity were also found in the stomach and duodenum of rats. In all cases, tissue extracts showed parallelism with reference PP (bovine) in the RIA. Gel chromatography (Sephadex G-50sf) of tissue extracts (rat, turtle) demonstrated a major peak of PP immunoreactivity, which eluted in the region of the reference PP. Salamander PP immunoreactivity eluted after bovine PP. In addition, the CTPP RIA can be applied to measure plasma levels of PP in rats, dogs, and humans. By using this PP RIA, we observed that plasma PP levels increase significantly in dogs (P less than 0.05) after intravenous administration of neurotensin. In rats, administration of intravenous bombesin resulted in a significant elevation of plasma PP.     

Identification of the sea urchin egg receptor for sperm using an antiserum raised against a fragment of its extracellular domain

Sea urchin egg fertilization requires the species-specific interaction of molecules on the sperm and egg surfaces. Previously, we isolated an extracellular, 70-kD glycosylated fragment of the S. purpuratus egg receptor for sperm by treating the eggs with lysylendoproteinase C (Foltz, K. R., and W. J. Lennarz. 1990. J. Cell Biol. 111:2951-2959). To characterize the receptor further, we have generated a polyclonal antiserum (anti-70KL) against the purified 70-kD fragment. Anti-70KL was found to react with a single polypeptide of approximately 350 kD on Western blots, presumed to be the intact receptor, in an egg cell surface preparation. This polypeptide appeared to be tightly associated with the plasma membrane/vitelline layer complex, as it was released from these preparations only by detergent treatment. Immunofluorescence microscopy revealed that the receptor was distributed evenly over the egg surface. The anti-70KL was species specific both in its ability to recognize the egg surface protein and to inhibit sperm binding. Fab fragments generated from affinity-purified anti-70KL also bound to the egg surface and inhibited sperm binding in a concentration-dependent manner. Interestingly, treatment with Fabs caused a small percentage of eggs to undergo cortical granule exocytosis, even in the absence of external Ca2+. These results confirm earlier findings indicating that the receptor is a cell surface glycoprotein of high molecular weight that species specifically binds sperm. This antiserum provides a powerful tool for further investigation of gamete interactions and the structure of the sperm receptor.     

Immunological detection of phytoene desaturase in algae and higher plants using an antiserum raised against a bacterial fusion-gene construct

Immunological characterization of phytoene desaturase, a key enzyme of carotenoid biosynthesis, is reported. For this purpose, a phytoene-desaturase fusion protein has been employed. For its construction 921 base pairs of the crtI gene were fused to the N-terminal region of the Escherichia coli lacZ gene. Plasmid pGABX2 resulted from insertion of a BglI - XhoI fragment from the Rhodobacter capsulatus carotenoid biosynthesis gene cluster, carrying the crtI, crtA and crtB genes, into pBR322. A 968-base-pair SalI-fragment from pGABX2 was cloned into pUR288 at the 3' end of the lacZ gene. Isopropyl-beta-D-thio-galactopyranoside-dependent activation of the lacZ fusion gene resulted in expression of a stable 150-kDa protein. After electroelution from SDS/polyacrylamide slab gels, the protein was used for antibody production. The heterogenic antiserum obtained was tested by Western blotting against proteins from Rhodobacter capsulatus and several different photoautotrophic organisms including higher plants. Apparent molecular masses of immunoreactive proteins from Rhodobacter, Aphanocapsa, rape and spinach were around 64 kDa. In Bumilleriopsis a 55-kDa protein was found instead. The antibody also inhibited in vitro desaturation of phytoene when detergent-solubilized membranes were employed.     

A radioimmunoassay for corticosterone and its application to the measurement of stress in poultry.

A radioimmunoassay for corticosterone was developed using an antibody to corticosterone-21-hemisuccinate:bovine serum albumin. The assay possessed good specificity, sensitivity and reproducibility and required minimal sample preparation. Tests of adrenal function showed that stimulation of the adrenal with exogenous ACTH and with dexamethasone caused an increase and decrease, respectively, in plasma concentrations of corticosterone. Exposure to cold environmental temperatures caused an increase in plasma corticosterone. Handling and the removal of blood samples by venepuncture had no effect upon the concentration of corticosterone. It was concluded that this assay would accurately measure the response to stresses which affect the pituitary-adrenal axis.     

Characterization and application of aptamers for Taq DNA polymerase selected using an evolution-mimicking algorithm

Using an evolution-mimicking algorithm (EMA), we have recently identified DNA aptamers that inhibit Taq DNA polymerase. In the present study, we have attempted to improve further the inhibitory activities of aptamers, as well as to characterize those aptamers with the most potent inhibitory activities. To characterize the most potent aptamer and demonstrate its applicability, the abilities to inhibit Tth DNA polymerase and to modulate specific amplification in PCR were investigated. This aptamer inhibited both Tth DNA polymerase and Taq DNA polymerase and improved the specificity of detection of a low-copy-number target gene in PCR using these DNA polymerases.     

Rapid and efficient isolation of highly specific antibodies from an antiserum against a pool of proteins

Antibodies with desired specificity to proteins of interest provide important and versatile tools for detecting and localizing specific proteins in organisms. With the rapidly increasing number of genes cloned, the demand for antibodies to the gene products is increasing greatly. We developed a procedure to isolate highly specific antibodies to an insect intestinal mucin (IIM) from a polyclonal antiserum, which served as a “library of antibodies,” by using an E. coli lysate of the IIM cDNA clone. This procedure allows rapid and efficient isolation of target protein specific antibodies from a polyclonal antiserum made against a pool of antigens.     

Phosphate-starvation-induced outer membrane proteins of members of the families Enterobacteriaceae and Pseudomonodaceae: demonstration of immunological cross-reactivity with an antiserum specific for porin protein P of Pseudomonas aeruginosa

Bacteria from members of the families Enterobacteriaceae and Pseudomonadaceae were grown under phosphate-deficient (0.1 to 0.2 mM Pi) conditions and examined for the production of novel membrane proteins. Of the 17 strains examined, 12 expressed a phosphate-starvation-induced outer membrane protein which was heat modifiable in that after solubilization in sodium dodecyl sulfate at low temperature the protein ran on gels as a diffuse band of higher apparent molecular weight, presumably an oligomer form, which shifted to an apparent monomer form after solubilization at high temperature. These proteins fell into two classes based on their monomer molecular weights and the detergent conditions required to release the proteins from the peptidoglycan. The first class, expressed by species of the Pseudomonas fluorescens branch of the family Pseudomonadaceae, was similar to the phosphate-starvation-inducible, channel-forming protein P of Pseudomonas aeruginosa. The second class resembled the major enterobacterial porin proteins and the phosphate-regulated PhoE protein of Escherichia coli. Using a protein P-trimer-specific polyclonal antiserum, we were able to demonstrate cross-reactivity of the oligomeric forms of both classes of these proteins on Western blots. However, this antiserum did not react with the monomeric forms of any of these proteins, including protein P monomers. With a protein P-monomer-specific antiserum, no reactivity was seen with any of the phosphate-starvation-inducible membrane proteins (in either oligomeric or monomeric form), with the exception of protein P monomers. These results suggest the presence of conserved antigenic determinants only in the native, functional proteins.     

A specific radioimmunoassay for PGE2 using an antibody with high specificity and a sephadex LH-20 microcolumn for the separation of prostaglandins.

Antiserum against PGE2 was raised in rabbits following immunization with prostaglandin-hen-gamma-globulin conjugate. The antiserum exhibited 14% cross reactivity with PGE1 and far less cross-reaction with heterologous prostaglandins. A microcolumn of Sephadex LH-20 was used for a partial, but sufficient separation of PGE2 from PGE1 and a complete separation from heterologous prostaglandins to ensure a specific RIA for PGE2. The precision of the method in the rage 10-500 picograms showed a coefficient of variation varying between 4 and 13%. The detection limit was 10 picograms corresponding to 15 pg/ml of PGE2 in serum. In order to demonstrate the validity of the method values obtained for non-diuretic rat renal venous serum were compared with those obtained using the isotope derivative method of Bojesen & Buckhave (1972) on the same samples. The concentrations of PGE2 obtained were 239 +/- 25 pg/ml and 250 +/- 58 pg/ml, respectively.     

Segmental peptidergic innervation of abdominal targets in larval and adult dipteran insects revealed with an antiserum against leucokinin I

Summary An antiserum against the cockroach neuropeptide leucokinin I (LKI) was used to study peptidergic neurons and their innervation patterns in larvae and adults of three species of higher dipteran insects, the flies Drosophila melanogaster, Calliphora vomitoria, and Phormia terraenovae, as well as larvae of a primitive dipteran insect, the crane fly Phalacrocera replicata. In the larvae of the higher dipteran flies, the antiserum revealed three pairs of cells in the brain, three pairs of ventro-medial cells in the subesophageal ganglion, and seven pairs of ventro-lateral cells in the abdominal ganglia. Each of these 14 abdominal leucokinin-immunoreactive (LKIR) neurons innervates a single muscle of the abdominal body wall (muscle 8), which is known to degenerate shortly after adult emergence. Conventional electron microscopy demonstrates that this muscle is innervated by at least one axon containing clear vesicles and two axons containing dense-cored vesicles. Electronmicroscopical immunocytochemistry shows that the LKIR axon is one of these two axons with dense-cored vesicles and that it forms terminals on the sarcolemma of its target muscle. The abdominal LKIR neurons appear to survive metamorphosis. In the adult fly, the efferent abdominal LKIR neurons innervate the spiracles, the heart, and neurohemal regions of the abdominal wall. In the crane fly larva, dorso-medial and ventrolateral LKIR cell bodies are located in both thoracic and abdominal ganglia of the ventral nerve cord. As in the larvae of the other flies, the abdominal ventrolateral LKIR neurons form efferent axons. However, in the crane fly larva there are two pairs of efferent LKIR neurons in each of the abdominal ganglia and their peripheral targets include neurohemal regions of the dorsal transverse nerves. An additional difference is that in the crane fly, a caudal pair of LKIR axons originating from the penultimate pair of dorso-median LKIR cells in the terminal ganglion innervate the hindgut.     

Human T lymphocyte receptor for sheep erythrocytes: Characterization of a specific antiserum and its application in the detection and quantitation of the receptor in a soluble form

An anti-human T lymphocyte serum specific to the receptor for sheep erythrocytes (E) was produced by immunizing sheep with the complex autologous E-soluble E receptor (ER_s). The soluble receptor (R_s) was obtained by heating human lymphocytes at 45 °C for 1 hr. The anti-R_s serum has been shown to inhibit E-rosette formation, to be cytotoxic to T cells, to identify T lymphocytes by indirect immunofluorescence, and to stimulate blastogenesis. The reaction of anti-R_s with R_s was directly demonstrated by two newly developed methods: agglutination of complexes formed by the treatment of formolized E with R_s (EFR_s complexes) and adhesion of a protein A producer strain of Staphylococcus aureus to EFR_s treated with anti-R_s. The anti-R_s antibodies could be neutralized by R_s present in supernatant of heated peripheral lymphocytes, inhibiting the above reactions and therefore providing methods to quantitate R_s in biological preparations. The importance of these assays is that R_s plays an immunoregulatory activity, and high levels of R_s in serum are associated with depressed cell-mediated immunity.     

Partial cloning and production of polyclonal antiserum against recombinant capsid protein of Hepatopancreatic Parvovirus (HPV) and its application for diagnostics in penaeid shrimp

Hepatopancreatic Parvovirus (HPV) causes infection in the early stages of shrimp leading to retarded growth, ultimaltely resulting in monetary loss to the shrimp farmers. To over come this situation screening of post-larvae (PL) by immunology-based diagnostics is required. Hence, the specific gene of capsid protein for HPV was cloned into pRSET B expression vector and rHCP overexpressed with 6-histidine tagged fusion protein in Escherichia coli BL21(DE3). Immunology-based methods like Western blot, dot blot and ELISA techniques were employed to detect HPV in infected samples using the antiserum raised in rabbits against recombinant HCP of HPV. The dot blot assay using anti-rHCP was found to be capable of detecting HPV in HPV infected post-larvae as early as at 24 h post infection. The antiserum could detect the HPV in the infected samples at 1 ng of total protein. HPV infection estimated by ELISA using anti-HCP and pure r-HCP as a standard was found to increase gradually during the course of infection from 24 h post infection. The sensitivity of antibody-based diagnostics employed in the present study was compared with that of PCR diagnostic method to screen the post-larvae for the detection of HPV.     

Characterization and application of an optical sensor for quantification of dissolved O2 in shake-flasks

On-line measurement of dissolved O₂ in shake-flasks was realized via immobilized sensor spots containing a fluorophore with an O₂-dependent luminescent decay time. An unaffected sensor signal during 80 autoclaving cycles suggests multi-usage of sensor equipped shake-flasks. The sensor had a response time of 6 s. Quantification of gas-liquid mass transfer revealed maximum k_La values of 150 h^–1, from which maximum O₂ transfer capacity of 33 mM h^–1 was calculated. Liquid volume and shaking frequency have a strong influence on k_La. Exemplified by cultivations of Corynebacterium glutamicum the importance of shaking rate for O₂ supply of bacterial cultures is shown. Sampling of microbial cultures with intermittent shaking of a few minutes can cause O₂ limitation. Based on the results of this work a simple and straightforward tool is now available for accurate O₂ sensing in shake-flasks, which are widely used in microbial cultivations.     

Immunocytochemical visualization of the Golgi apparatus in several species, including human, and tissues with an antiserum against MG-160, a sialoglycoprotein of rat Golgi apparatus

We used a monoclonal antibody (10A8), derived from mice immunized with fractions enriched in Golgi apparatus of rat brain neurons, to isolate an intrinsic membrane sialoglycoprotein of 160 KD from rat brain. By immunoelectron microscopy the sialoglycoprotein, named MG-160, was localized in medical cisternae of the Golgi apparatus of neurons, glia, adenohypophysis, and cultured rat pheochromocytoma (PC 12). The monoclonal antibody (MAb) reacted only with rat tissues. Because the epitope(s) recognized by a monoclonal antibody may be restricted, localization of an antigen by a single MAb may not reflect the extent of the distribution of antigen in various species and tissues. Therefore, to further investigate the presence and localization of MG-160 or of an antigenically related protein in several species and tissues, we used a polyclonal antiserum raised against MG-160 purified by antibody (10A8) affinity chromatography. Immunoblots of crude microsomal fractions from rat brain probed with the antiserum against MG-160 showed two to three prominent bands of approximately 160, 150, and 68 KD. Immunoblots of crude microsomal fractions from human, chicken, and frog brains showed prominent bands of 130-140 and 68 KD. Immunoblots of crude membrane fractions from Saccharomyces cerevisiae showed prominent bands of approximately 110-120 and 80 KD. Light microscopic immunocytochemical studies with frog, chicken, mouse, rat, rabbit, bovine, and human brains and with several other rat and human tissues showed a staining pattern consistent with the Golgi apparatus. Immunoelectron microscopy with rat and human brain and with rat myocardium and pituitary showed prominent and exclusive staining of cis, medial, and occasionally trans cisternae of the Golgi apparatus. The cisternae of the trans Golgi network were not stained. These findings are consistent with the hypothesis that a polypeptide related to MG-160 is present in the Golgi apparatus of several tissues in human, rodents, chicken, and frog and possibly in Saccharomyces cerevisiae. The antiserum to MG-160 represents a reliable reagent for immunohistochemical visualization of the Golgi apparatus in brain and several other human tissues obtained at autopsy, fixed with Bouin's, and embedded in paraffin.     

Algorithms for extracting structured motifs using a suffix tree with an application to promoter and regulatory site consensus identification.

This paper introduces two exact algorithms for extracting conserved structured motifs from a set of DNA sequences. Structured motifs may be described as an ordered collection of p > or = 1 "boxes" (each box corresponding to one part of the structured motif), p substitution rates (one for each box) and p - 1 intervals of distance (one for each pair of successive boxes in the collection). The contents of the boxes--that is, the motifs themselves--are unknown at the start of the algorithm. This is precisely what the algorithms are meant to find. A suffix tree is used for finding such motifs. The algorithms are efficient enough to be able to infer site consensi, such as, for instance, promoter sequences or regulatory sites, from a set of unaligned sequences corresponding to the noncoding regions upstream from all genes of a genome. In particular, both algorithms time complexity scales linearly with N2n where n is the average length of the sequences and N their number. An application to the identification of promoter and regulatory consensus sequences in bacterial genomes is shown.