An immunoenzyme method of isolation of foot-and-mouth disease virus by using beta-lactamase conjugate with virus-specific antibodies

It was shown that in was feasible to use conjugates of virus-specific antibodies and beta-lactamase from Bacillus licheniformis 749/c to identify aphthosa virus antigens. The antigen titers determined by enzyme immunoassay (EIA) using a beta-lactamase conjugate were 5-64 times higher than the analogous indices of the complement fixation test. Unlike EIA, that by using the antibody conjugates with peroxidase or alkaline phosphatase there were observed no "background" responses.     

Synthesis of 5'-oligonucleotide hydrazide derivatives and their use in preparation of enzyme-nucleic acid hybridization probes

A hydrazone-based method for conjugating synthetic nucleic acids and reporter molecules for use as nonradioactive hybridization probes is presented. Oligonucleotides complementary to the hepatitis B virus were derivatized at their 5' ends with hydrazine or homobifunctional acyl hydrazides. These derivatives reacted facilely with aldehydes to give hydrazones, which were characterized by uv spectroscopy and HPLC. Coupling of aldehyde-modified alkaline phosphatase with carbohydrazide-oligonucleotide derivatives provided a mixture of two enzyme-nucleic acid conjugates in 80-85% yield. The conjugates had a 1:1 and a 2:1 oligonucleotide/enzyme ratio, respectively, and were separated by ion-exchange chromatography. Both conjugates were able to detect 7 amol of target DNA in 1 h, using a colorimetric assay. In contrast, oligonucleotide-horseradish peroxidase conjugates were 40-fold lower in sensitivity of detection.     

An improved preparation and purification of oligonucleotide-alkaline phosphatase conjugates.

A simple, low-cost protocol giving good yields of oligonucleotide-alkaline phosphatase conjugates on a 7-nmol or a 35-nmol scale of oligomer has been developed. The cross-linking agent is disuccinimidyl suberate. n-Butanol is used to remove excess disuccinimidyl suberate and side products away from the disuccinimidyl suberate/oligomer adduct before alkaline phosphatase is added directly to the dried adduct. The crude conjugate is purified in one step using a DEAE HPLC column and an NaCl gradient. These conjugates were used to detect 0.4 pg of a hepatitis B virus sequence using a chemiluminescent assay.     

DNA hybridization assay using ATTOPHOS, a fluorescent substrate for alkaline phosphatase.

A fluorometric procedure for the detection of DNA-DNA hybrids is described. The procedure involved the detection of probe-bound alkaline phosphatase with the fluorescent substrate ATTOPHOS. This substrate is converted to ATTOFLUOR by alkaline phosphatase and fluoresces strongly at 550 nm when excited with a wavelength of 440 nm. DNA hybridization assays were performed both with dilutions of purified target plasmid DNA (pSE9 or PBR322) and whole bacterial cells. Streptavidin-alkaline phosphatase conjugates were added to react with bound probe. Fluorometric assays, as well as colorimetric assays, using 5-bromo-4-chloro-3-indolylphosphate + nitroblue tetrazolium for alkaline phosphatase activity were performed. The fluorescence of the substrate was measured at time intervals, and the slope of the regression line calculated. A slope four times greater than that of background was considered positive. One hundred femtograms or 2.2 x 10(4) molecules of homologous DNA were detected with the fluorescent assay as compared with 10,000 femtograms or 2.2 x 10(6) molecules of homologous DNA with the colorimetric assay. Similar results were obtained with whole cells. Approximately 1 x 10(3) homologous cells were detected fluorometrically and 1 x 10(5) cells were detected colorimetrically. Based on these results, we conclude that, in our hands, the DNA hybridization assay described here using ATTOPHOS as the substrate for alkaline phosphatase is a very sensitive assay for the detection of DNA-DNA hybrids.     

Evaluation of the use of chromogenic and fluorogenic substrates in solid-phase enzyme linked immunosorbent assays (ELISA)

Fluorogenic and chromogenic substrates were used in direct and trapping enzyme-linked immunosorbent assays (ELISA) for the detection of mouse IgG and foot-and-mouth disease virus (FMDV). The detection limits for both antigens were compared using different combinations of enzymes and substrates. Various times and concentrations of chemicals were used to obtain maximum sensitivity for both systems. Similar sensitivities were found using fluorogenic and chromogenic substrates. Tetramethyl benzidine substrate for horse-radish peroxidase enzyme conjugates was found to attain the highest sensitivity levels for chromogenic assays (0.12 ng IgG/ml and 1.0 ng/ml FMDV respectively), after 10 min incubation. Of the two fluorogenic enzyme/substrates studied, B-galactosidase was the most sensitive but required extended incubation times (2-3 h) as compared with chromogenic systems. Special microplates for fluoro-immunoassay (FIA) were compared with conventional microplates and no advantage was found to justify their use. An alkaline phosphatase anti-guinea-pig conjugate was used to confirm the equivalence of fluorogenic and chromogenic substrates in terms of sensitivity. A comparison of the amount of signal generated using various concentrations of enzyme in the absence of antigen was made for two different alkaline phosphatase conjugates to obtain theoretical sensitivity limits. One possible advantage of fluorogenic substrates is that high binding ratio can improve the confidence in discrimination of positive results.     

A comparison of chemiluminescent and colorimetric substrates in a hepatitis B virus DNA hybridization assay

We compared the sensitivity of a chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD) and a chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) for detection of an alkaline phosphatase label in a hepatitis B virus "core antigen" DNA (HBVc) probe hybridization assay. Chemiluminescent signal obtained from AMPPD hydrolysis by alkaline phosphatase was detected with Polaroid Instant Black and White Type 612 film. The chemiluminescent assay detected 1.18 x 10(6) copies of HBVc plasmid DNA in 30 min. By comparison, 9.8 x 10(7) copies of DNA could be measured using chromogenic BCIP/NBT substrate within the same incubation time. After further development, the chemiluminescent endpoint permitted detection of 4.39 x 10(4) copies of HBVc plasmid DNA in 2 h.     

基于纳米金复合探针的基因芯片膜转印检测法

建立了一种基于纳米金复合探针的基因芯片膜转印核酸检测新方法。首先,用纳米金颗粒同时标记检测探针P2和两种长短不同且生物素化的信号探针 (T10,T40),其中检测探针与靶DNA 5￠端互补,两种信号探针起信号放大作用。当靶DNA分子存在时,芯片表面捕捉探针P1 (与靶DNA分子3￠端互补) 通过碱基互补配对原则结合靶DNA分子,将其固定于芯片上,同时检测探针通过与靶DNA 5￠端互补配对将纳米金复合探针结合于芯片表面,结果在芯片表面形成“三明治”结构,后通过链霉亲和素-生物素反应,使芯片表面对应有靶DNA分子的部位结合上碱性磷酸酶,最后利用BCIP/NBT显色系统使芯片表面信号结果镜面转印至尼龙膜表面。当检测探针和信号探针摩尔比为1∶10,T10和T40摩尔比为9:1时可以检测1 pmol/L合成靶DNA分子或0.23 pmol/L结核分枝杆菌16S rDNA PCR扩增产物,检测结果通过普通的光学扫描仪读取或肉眼直接判读信号有无。本芯片检测系统灵敏度高,操作方法简单、快速,不需要特殊仪器设备,在生物分子的检测方面具有较高的应用价值。     

Preparation of enzyme-conjugated DNA probe and application to the universal probe system.

A general procedure for the cross-linking of enzyme to DNA has been developed for use as a nonradioactive probe. In this method, DNA is transaminated with diaminopropane to introduce primary amino groups into the cytosine residues. Then the amino groups are converted to thiol groups using a heterobifunctional cross-linker. The thiolated DNA is conjugated with the maleimide-introduced enzyme. With this method, alkaline phosphatase was cross-linked to a single-stranded DNA (sspUCRf1). The conjugate was able to detect 5 pg of target DNA (pUCf1 plasmid, 3.2 kbp) fixed onto the nitrocellulose membrane, using a colorimetric assay. The enzyme-conjugated DNA was applied to "the universal probe system," which consisted of two single-stranded DNA probes (a primary probe and a labeled secondary probe). Using alkaline phosphatase-conjugated sspUCRf1 DNA as the secondary probe, the c-myc gene and HBV DNA were detected effectively on Southern and dot-blot hybridization.     

耐热性碱性磷酸酯酶作为非放射性标记物的研究

通过生物素与亲和素－酶复合物系统或地高辛与抗地高辛－酶复合物系统可把酶间接标记到探针上．Ｒｅｎｚ等通过不同的化学方法直接把酶标记到探针上［１～３］．耐热性碱性磷酸酯酶ＦＤ－ＴＡＰ（ｔｈｅｒｍｏｓｔａｂｌｅａｌｋａｌｉｎｅｐｈｏｓｐｈａｔａｓｅ）具有耐...     

Structural insights into the conformation and oligomerization of E2~ubiquitin conjugates

Post-translational modification of proteins by ubiquitin (Ub) regulates a host of cellular processes, including protein quality control, DNA repair, endocytosis, and cellular signaling. In the ubiquitination cascade, a thioester-linked conjugate between the C-terminus of Ub and the active site cysteine of a ubiquitin-conjugating enzyme (E2) is formed. The E2~Ub conjugate interacts with a ubiquitin ligase (E3) to transfer Ub to a lysine residue on a target protein. The flexibly linked E2~Ub conjugates have been shown to form a range of structures in solution. In addition, select E2~Ub conjugates oligomerize through a noncovalent "backside" interaction between Ub and E2 components of different conjugates. Additional studies are needed to bridge the gap between the dynamic monomeric conjugates, E2~Ub oligomers, and the mechanisms of ubiquitination. We present a new 2.35 ? crystal structure of an oligomeric UbcH5c~Ub conjugate. The conjugate forms a staggered linear oligomer that differs substantially from the "infinite spiral" helical arrangement of the only previously reported structure of an oligomeric conjugate. Our structure also differs in intraconjugate conformation from other structurally characterized conjugates. Despite these differences, we find that the backside interaction mode is conserved in different conjugate oligomers and is independent of intraconjugate relative E2-Ub orientations. We delineate a common intraconjugate E2-binding surface on Ub. In addition, we demonstrate that an E3 CHIP (carboxyl terminus of Hsp70 interacting protein) interacts directly with UbcH5c~Ub oligomers, not only with conjugate monomers. These results provide insights into the conformational diversity of E2~Ub conjugates and conjugate oligomers, and into their compatibility and interactions with E3s, which have important consequences for the ubiquitination process.     

A multiple-staining procedure for the detection of different DNA fragments on a single blot.

We have developed a method that implies the use of a particular type of substrate which can be used in combination with alkaline phosphatase in detecting nucleic acid on filters. The method allows the detection of several different nucleic acid sequences on a single filter. In consecutive steps, the target DNA molecules are hybridized with different digoxigenin-labeled DNA probes. After each hybridization step, digoxigenin is detected with an antibody-alkaline phosphatase conjugate. This enzyme is subsequently visualized by a color reaction using different 2-hydroxy-3-naphthoic acid anilide (naphthol AS) phosphates as substrates in combination with varying diazonium salts. The multiple-staining procedure is based on the fact that the probe DNA-antibody complex can be removed while the color precipitate remains stably bound at its place on the filter. This allows several repeated hybridizations with other digoxigenin-labeled probes followed by antibody detection and color reaction with other naphthol AS phosphate-diazonium salt combinations. Aside from the ability to simultaneously visualize different target DNAs on a single filter, this new method provides several important features that are more powerful than the conventional 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium (BCIP-NBT) color reaction for alkaline phosphatase. The colors are more stable and brilliant than BCIP-NBT; their development is faster, the resolution of closely spaced bands is greater, and the background is much lower. The detection limit for alkaline phosphates is as good as with BCIP-NBT (0.1 pg of DNA). One major advantage is the simplicity of removing the colors by ethanol incubation. In this paper, the method is described using the example of Southern blotted DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stress Proteins and Isozymology of Acid Phosphatase, Esterase, and Peroxidase in Phaseolus vulgaris Following Peanut Mottle Virus Infection

The present work was undertaken to study soluble protein changes and enzyme alterations in "Topcrop" bean ( Phaseolus vulgaris L.) primary leaves following inoculation with peanut mottle virus (PMV) in an attempt to elucidate the biochemical basis of the hypersensitive reaction in this host virus combination. Using the discontinuous polyacrylamide gel electrophoresis (disc-PAGE) technique, at least four apparently "novel" protein bands at Rf values of 0.80, 0.77, 0.74 and 0.68 were observed in infected tissue. The band at Rf 0.76 seems to be injury related and is shown here to be an isozyme of acid phosphatase. It is believed that these proteins are neither the cause nor the product of necrosis and may thus play a role in the active defense mechanism of the plant. In this virus host combination it was found that heating soluble protein fractions at 55 °C for 10 min before electrophoresis dramatically reduced the background and improved resolution on gels. While the biological function(s) of these proteins needs further investigations it is almost certain that none of them is an isozyme of alkaline phosphatase, acid phosphatase, esterase, or peroxidase. Zymogram analysis has additionally revealed the absence of alkaline phosphatase activity in untreated, abraded and PMV-infected primary leaves and no qualitative or quantitative changes in esterase isozymes have been observed in primary leaf tissues following abrasion or PMV–infection. By contrast, qualitative alterations in acid phosphatase after PMV-infection and both qualitative and quantitative changes in peroxidase after mechanical abrasion and PMV-infection have also been demonstrated.     

Properties of alkaline phosphatase in crude homogenates of human diploid fibroblasts

The kinetic properties of the "constitutive" and the "induced" alkaline phosphatase in diploid fibroblasts are compared with those of the enzymes in crude tissue homogenates. Both the constitutive as the induced enzyme have properties comparable with those of the liver-bone-kidney group. The induced alkaline phosphatase clearly differs from the "constitutive" alkaline phosphatase concerning the effect of high concentrations of L-phenylalanine and the effect of Mg2+ ions. The induced alkaline phosphatase seems to be identical with the enzyme in liver, but the constitutive alkaline phosphatase could not be identified.     

Induction of placental alkaline phosphatase in choriocarcinoma cells by 5-bromo-2′-deoxyuridine

Summary Growth of choriocarcinoma cells in the presence of 5-bromo-2′-deoxyuridine (BrdUrd) results in a 30- to 40-fold increase in alkaline phosphatase activity. The effect of BrdUrd is specific for phosphatase with an alkaline pH optimum. The induction by BrdUrd is probably not due to the production of an altered enzyme, since the induced enzyme resembles the basal enzyme in thermal denaturation and kinetic properties. Enzyme induction can be prevented by thymidine but not by deoxycytidine or deoxyuridine. The induction of alkaline phosphatase appears to require incorporation of the BrdUrd into cellular DNA. The presence of BrdUrd in the growth medium is not necessary for alkaline phosphatase induction in proliferating cells containing: BrdUrd-substituted genomes. However, enzyme induction and maintenance of the induced levels of alkaline phosphatase in nonproliferating cells containing BrdUrd-substituted DNA requires the presence of the analogues in the medium. The induction of alkaline phosphatase by BrdUrd in probably an indirect process.     

Induction of placental alkaline phosphatase in choriocarcinoma cells by 5-bromo-2'-deoxyuridine.

Growth of choriocarcinoma cells in the presence of 5-bromo-2'-deoxyuridine (BrdUrd) results in a 30- to 40-fold increase in alkaline phosphatase activity. The effects of BrdUrd is specific for phosphatase with an alkaline pH optimum. The induction by BrdUrd is probably not due to the production of an altered enzyme, since the induced enzyme resembles the basal enzyme in thermal denaturation and kinetic properties. Enzyme induction can be prevented by thymidine but not by deoxycytidine or deoxyuridine. The induction of alkaline phosphatase appears to require incorporation of the BrdUrd into cellular DNA. The presence of BrdUrd in the growth medium is not necessary for alkaline phosphatase induction in proliferating cells containing BrdUrd-substituted genomes. However, enzyme induction and maintenance of the induced levels of alkaline phosphatase in nonproliferating cells containing BrdUrd-substituted DNA requires the presence of the analogues in the medium. The induction of alkaline phosphatase by BrdUrd in probably an indirect process.     

Conjugate formation in urea: coupling of insoluble peptides to alkaline phosphatase for ELISA and in situ detection of antibody-forming cells

We report here a new method to produce synthetic peptide/alkaline phosphatase (AP) conjugates in the presence of urea. The method allows the use of peptides that are not soluble to a sufficient degree in aqueous buffers. The presence of 8 M urea during the construction of the synthetic peptide/AP conjugates does not influence enzyme activity nor the affinity of the anti-peptide antibodies for the conjugated peptide. We demonstrate that these synthetic peptide/AP conjugates can be used for detection of specific antipeptide antibody-forming cells (AFC) in vivo. This method for constructing enzyme conjugates with insoluble proteins or peptides suggest not only new possibilities for detection of specific AFC in vivo but also for applications in receptor-ligand studies, ELISA (enzyme-linked immunosorbent assay), and spot ELISA for detection of antibody-secreting cells in vitro.     

Effect of pentagastrin, secretin and cholecystokinin on growth and differentiation in organ cultured rabbit small intestine

The effect of pentagastrin, secretin and cholecystokinin on biochemical parameters of mucosal growth and differentiation was studied in organ cultured rabbit jejunum and ileum. Pentagastrin at 0.05-5.0 microgram/ml did not affect DNA content of the biopsy, but led to a significant decrease of sucrase and alkaline phosphatase activity in the ileum. Secretin prompted a significant decrease of DNA and protein in the ileum at a level of 10(-7) and 10(-5) M, but had no effect in the jejunum. Of the brush border enzymes, sucrase and alkaline phosphatase were suppressed in both parts of the intestine both with respect to specific activity and total biopsy content. Cholecystokinin, like pentagastrin, did not influence DNA or protein content, but reduced sucrase, maltase and alkaline phosphatase activity. HMG-CoA reductase, the key enzyme of cholesterol synthesis, was not significantly affected by any of the three hormones tested. When brush border enzymes or DNA from desquamated cells were measured in the post-culture medium, no consistent effect of any gastrointestinal hormone was apparent. The present study demonstrates a direct "antitrophic" effect of secretin in cultured mucosa. Pentagastrin and cholecystokinin did not influence mucosal DNA content in vitro but apparently inhibited villus cell differentiation.     

MagSNiPer: a new single nucleotide polymorphism typing method based on single base extension, magnetic separation, and chemiluminescence

We have developed a new method for typing single nucleotide polymorphisms (SNPs), MagSNiPer, based on single base extension, magnetic separation, and chemiluminescence. Single base nucleotide extension reaction is performed with a biotinylated primer whose 3' terminus is contiguous to the SNP site with a tag-labeled ddNTP. Then the primers are captured by magnetic-coated beads with streptavidin, and unincorporated labeled ddNTP is removed by magnetic separation. The magnetic beads are incubated with anti-tag antibody conjugated with alkaline phosphatase. After the removal of excess conjugates by magnetic separation, SNP typing is performed by measuring chemiluminescence. The incorporation of labeled ddNTP is monitored by chemiluminescence induced by alkaline phosphatase. MagSNiPer is a simple and robust SNP typing method with a wide dynamic range and high sensitivity. Using MagSNiPer, we could perform SNP typing with as little as 10(-17) mol of template DNA.     

Production of fetal-like alkaline phosphatase by HeLa cells

Alkaline phosphatase produced by HeLa cells differs in its chemical and physical properties from the enzyme found in adult organs and tissues (Cox and Griffin, 1967). In the present study HeLa cell alkaline phosphatase was compared to a fetal form of the enzyme found in human placenta. Both enzymes have approximately the same molecular weight as judged by sucrose density gradients, and the chemical and physical properties of these alkaline phosphatases are similar. The electrophoretic pattern of the HeLa cell enzyme resembles the placental alkaline phosphatase of the heterozygous FS phenotype except that it is slower moving. Double immunodiffusion using an antibody against HeLa cell alkaline phosphatase and placental and HeLa cell enzymes as antigens shows a single line of partial identity between the two enzymes, with a small spur suggesting additional antigenic sites on the HeLa cell enzyme. The data suggest that malignant cells in culture, HeLa, are producing a fetal-like alkaline phosphatase probably by derepression of the genome. However, the electrophoretic and immunological characteristics of the enzyme are altered sufficiently so that it can be distinguished from the normally produced fetal enzyme.This work was supported by U.S. Public Health Service Grant GM 15508 and the Health Research Council of the City of New York.Fourth-year student; Honors Program.Career Scientist Health Research Council of the City of New York.     

Immunocytological investigation of protein synthesis in Escherichia coli.

Ferritin-conjugated specific antibodies have been used to localize beta-galactosidase and both the monomer and active dimer of alkaline phosphatase in frozen thin sections of cells of Escherichia coli O8 strain F515. The even distribution of the ferritin marker throughout cells that had been induced for beta-galactosidase synthesis, frozen, sectioned, and exposed to ferritin-anti-beta-galactosidase conjugate showed that this enzyme was present throughout the cytoplasm of these cells. Frozen thin sections of cells that had been derepressed for the synthesis of alkaline phosphatase were exposed to both ferritin-anti-alkaline phosphatase monomer and ferritin-anti-alkaline phosphatase dimer conjugates, and the ferritin markers showed a peripheral distribution of both the monomer and the dimer of this enzyme. This indicates that alkaline phosphatase is present only in the peripheral regions of the cell and argues against the existence of a cytoplasmic pool of inactive monomers of this enzyme. This peripheral location of both the monomers and dimers of alkaline phosphatase supports the developing concensus that this enzyme is, like other wall-associated enzymes, synthesized in association with the cytoplasmic membrane and vectorially transported to the periplasmic area, where it assumes its tertiary and quaternary structure and acquires its enzymatic activity.