Immunochemical quantification of procion red HE-3B used as ligand in affinity chromatography.

The quantification of Procion Red HE-3B used as a ligand in affinity chromatography for proteins is reported. It's based on an enzyme-linked immunosorbent assay using antibodies against the dye. Polyclonal antibodies were classically prepared after conjugation of the dye on KLH and injection into rabbits. The development of the assay was based on the competitive inhibition between hemoglobin-dye complex and free dye. The sensitivity of this method was about 1000-times higher than a classical spectrophotometric assay, and was modulated by some chemical substituents attached on the native dye. It was demonstrated that the assay was applicable to the determination of dye traces that may be released from dye affinity sorbents. Moreover, the quantification of the dye was successfully applied to proteins that are being purified from a dye affinity column.     

HIV-1 Nef Breaches Placental Barrier in Rat Model

The vertical transmission of HIV-1 from the mother to fetus is known, but the molecular mechanism regulating this transmission is not fully characterized. The fetus is highly protected by the placenta, which does not permit microbial pathogens to cross the placental barrier. In the present study, a rat model was established to observe the effect of HIV-1 protein Nef on placental barrier. Evans blue dye was used to assay permeability of placental barrier and fourteen day pregnant Sprague Dawley rats were injected intravenously with 2% Evans blue dye along with various concentrations of recombinant Nef. After an hour, animals were sacrificed and dye migration was observed through the assimilation of peripheral blood into fetus. Interestingly, traces of recombinant Nef protein were detected in the embryo as well as amniotic fluid and amniotic membrane along with placenta and uterus. Our study indicates that recombinant HIV-1-Nef protein breaches the placental barrier and allows the migration of Evans blue dye to the growing fetus. Further the concentration of Nef protein in blood is directly proportional to the intensity of dye migration and to the amount of Nef protein detected in uterus, placenta, amniotic membrane, amniotic fluid and embryo. Based on this study, it can be concluded that the HIV-1 Nef protein has a direct effect on breaching of the placental barrier in the model we have established in this study. Our observations will be helpful to understand the molecular mechanisms related to this breach of placental barrier by Nef in humans and may be helpful to identify specific Nef inhibitors.     

Development of a Disperse Dye Immunoassay Technique for Detection of Antibodies against Neospora caninum in Cattle

In this study a disperse dye immunoassay method was standardized and evaluated for detection of antibodies against Neospora caninum in cattle. Sera from 150 cattle with a recent history of abortion were collected and tested by commercial ELISA kit and a standardized in-house dye immunoassay system. The positivity rate for the sera used in this study was 34.6% for the disperse dye immunoassay (DDIA) compared to 32% obtained by ELISA kit. This study showed no significant difference between DDIA and ELISA. The results indicated that the DDIA provide an economic, simple, rapid and robust test for detection of N. caninum infection in cattle.     

An investigation of bidirectional translocation in the Phloem

Patterns of (14) CO(2) , assimilate movement in Vicia jaba plants having 7 nodes were studied. Bidirectional translocation occurred throughout most of the stem length when tracer was applied to leaves of various ages. To determine whether this bidirectional translocation occurs within single sieve tubes, a O.1 % solution of the fluorescent dye K-fluorescein was applied to a lightly scraped area on the stem in the middle of a young internode. After one hour the dye was present short distances above and below the treated area. Free-hand sections of the internode showed the dye to be localized in the traces of the larger leaves below tbe treated area and in the traces of the younger leaves above the treated area. The dye was never present in the same bundle both above and below the treated area, indicating that each bundle and sieve tube translocated the dye in only one direction. These results were confirmed using Phaseolus vulgaris, Vinca rosea, and Pelargonium hortum. A similar study in which petioles of young Ecballium elaterium leaves were treated showed that usually the phloem of one bundle translocated the dye in only one direction but in some cases the external phloem of the bicollateral bundles carried the dye toward the stem while the internal phloem carried the dye toward the blade. When longer time intervals were used in all these experiments, the dye sometimes appeared in the same phloem areas both above and below the treated area. This is explained by a lateral transfer of tracer within the phloem, either through secondary phloem or through bundle anastomoses at the nodes.     

Detection of trace aflatoxin M1 in human urine using a commercial ELISA followed by HPLC

Aflatoxin is a liver carcinogen, and rapid, inexpensive methods to detect its urinary biomarkers are needed. We used a commercial enzyme-linked immuno-sorbent assay (ELISA) for aflatoxin M1 in urine (Helica Biosystems) to test 52 Haitian samples. Using this ELISA, we detected traces above the limit of detection (0.2?ng/ml urine) but below the limit of quantitation (0.4?ng/ml) in 14 samples. Liquid chromatography of all 52 Haitian urine samples revealed that only 11 had quantifiable AFM1 (mean: 29.5?pg/ml, standard error: 10.8, range: 2.94–96.5?pg/ml). The Helica ELISA may have detected forms of aflatoxin other than AFM1 in the Haitian samples, or matrix enhancement may have affected results at low AFM1 concentrations. This ELISA may serve as an initial, qualitative indicator of aflatoxin exposure for epidemiological purposes. But this method’s utility as a precise and specific indicator of AFM1 concentrations will require additional refinement and validation.     

Evaluation of serodiagnosis of toxoplasmosis by using the recombinant nucleoside triphosphate hydrolase isoforms expressed in Escherichia coli

The nucleoside triphosphate hydrolase (NTPase) isoforms termed, NTPase-I and NTPase-II of Toxoplasma gondii, were expressed in Escherichia coli as inclusion bodies and purified under denaturing condition. Furthermore, NTPase-I was refolded as an active form and purified under non-denaturing condition. The purified NTPase isoforms, both denatured and refolded, were tested for their usefulness as antigens for the serodiagnosis of acute toxoplasmosis in immunocompetent humans. The test was conducted by using the recombinant NTPase isoforms and comparing the enzyme linked immunosorbent assay (ELISA) absorbances with the Sabin-Feldman dye test titer. Seventy-three sera from dye test-positive patients, and 30 sera from subjects with no T. gondii infection were examined. The total positive rates in dye test positive sera were: 82% (60/73) for denatured NTPase-I; 78% (57/73) for denatured NTPase-II; and 63% (46/73) for refolded NTPase-I. For all three antigen types of recombinant NTPase, the positive rates of sera of acute toxoplasmosis suspected patients were 93% (13/14). A moderate correlation between the ELISA absorbance using these antigens and the dye test titer was observed with the correlation coefficients, 0.583 (r2) for denatured NTPase-I, 0.472 (r2) for denatured NTPase-II, and 0.604 (r2) for refolded NTPase-I in the linear regression analysis. There was no significant difference observed in the antigenicity between refolded and denatured NTPase-I, nor between the isoforms.     

α-Santalol, a derivative of sandalwood oil, induces apoptosis in human prostate cancer cells by causing caspase-3 activation

The anticancer effects of α-santalol, a major component of sandalwood oil, have been reported against the development of certain cancers such as skin cancer both in vitro and in vivo. The primary objectives of the current study were to investigate the cancer preventive properties of α-santalol on human prostate cancer cells PC-3 (androgen independent and P-53 null) and LNCaP (androgen dependent and P-53 wild-type), and determine the possible mechanisms of its action. The effect of α-santalol on cell viability was determined by trypan blue dye exclusion assay. Apoptosis induction was confirmed by analysis of cytoplasmic histone-associated DNA fragmentation using both an apoptotic ELISA kit and a DAPI fluorescence assay. Caspase-3 activity was determined using caspase-3 (active) ELISA kit. PARP cleavage was analyzed using immunoblotting. α-Santalol at 25-75 μM decreased cell viability in both cell lines in a concentration and time dependent manner. Treatment of prostate cancer cells with α-santalol resulted in induction of apoptosis as evidenced by DNA fragmentation and nuclear staining of apoptotic cells by DAPI. α-Santalol treatment also resulted in activation of caspase-3 activity and PARP cleavage. The α-santalol-induced apoptotic cell death and activation of caspase-3 was significantly attenuated in the presence of pharmacological inhibitors of caspase-8 and caspase-9. In conclusion, the present study reveals the apoptotic effects of α-santalol in inhibiting the growth of human prostate cancer cells.     

Efficient screening of high-signal and low-background antibody pairs in the bio-bar code assay using prion protein as the target

The bio-bar code assay is an assay for ultrasensitive detection of proteins. The main technical hurdle in bio-bar code assay development is achieving a dose-dependent, reproducible signal with low background. We report on a magnetic bead ELISA screening mechanism for characterizing antibody pairs that are effective for use in the bio-bar code assay. The normal isoform of prion protein was utilized as the target protein as dozens of antibodies have been developed against it. The development of an ultrasensitive assay for the detection of the various isoforms of PrP has the potential to enable significant advances in the diagnosis and understanding of transmissible spongiform encephalopathies, including transmission mechanisms, disease pathology, and potential therapeutics. With prion protein as the target, the magnetic bead ELISA identified pairs with high background and low signal in the bio-bar code assay. The magnetic bead ELISA was effective as a screening mechanism because it reduced assay time and cost and allowed for understanding of pair characteristics such as development times and signal-to-noise ratios.     

A phage-linked immunoabsorbant system for the detection of pathologically relevant antigens

This report describes a novel system for the immunological detection of immobilized antigen. The detection of herpes simplex virus (HSV) antigen was used as an example. Bacteriophage M13, containing the E. coli lac Z gene, was used as the "reporter" molecule in an immunoassay which is otherwise analogous to the enzyme-linked immunoabsorbant assay (ELISA). Briefly, HSV infected cells were incubated with a mouse monoclonal antibody specific for HSV antigen, followed by rabbit anti-mouse serum and mouse anti-M13 serum. Immune complexes were incubated with viable M13 phage. M13 binding was due to the presence of M13 antibodies, whose presence ultimately depended on the binding of monoclonal antibody to HSV. Phage was recovered by elution in pH = 11. Recovered phage was used to infect E. coli. M13 was quantitated by either plaque assay or by an assay for phage-induced beta-galactosidase activity in appropriate E. coli strains. The amount of M13 recovered was proportional to the number of HSV infected cells probed. Therefore, M13 served as a "bio-amplifiable tag" to antibody, as enzymes do in the ELISA. Since M13 is viable, its signal can be amplified by infection of susceptible bacteria, and the promise for an enormously sensitive immunoassay exists. The sensitivity of the assay described here is compared to the ELISA in the detection of HSV infection cells, as an example of the novel assay's potential. Significantly, the novel assay was more sensitive than the ELISA when samples were tested under identical circumstances. This technique is called the phage-linked immunoabsorbant assay (PHALISA), by analogy to the ELISA.     

Enzyme-immunoassay for the determination of metallothionein in human urine: application to environmental monitoring

The objectives of this study were to develop an enzyme immunoassay for metallothioneins in human urine using a polyclonal antiserum and to demonstrate a possible relationship between the level of this biomarker and heavy metal exposure. The antiserum was raised in sheep against horse metallothionein conjugated to carboxylated bovine serum albumin. The antibody was used to construct a two-step competitive ELISA procedure. Human urine was treated with activated charcoal powder to remove traces of metallothioneins and known amounts of pure metallothioneins were added to provide standards for a standard curve. Metallothionein levels were measured in two groups of children living in areas of mild and high environmental pollution due mainly to heavy metals. A comparison was made between the biomarker levels and the levels of cadmium and lead in urine samples in the two groups. A group of children from a non-polluted area acted as controls. The results show that the detected levels of metallothioneins appear to correspond to levels of the two heavy metals studied and that there was an apparent relationship to the environmental exposure. Thus according to results of this study the increase in the metallothionein excretion seems to provide an indication of previous of exposure to metals. The ELISA procedure is sensitive and robust and can be used to screen large numbers of samples and is more rapid than the physical procedures currently used for analysis of these proteins. The assay can therefore be used as an additional tool for screening at-risk populations where either environmental or occupational exposure to divalent heavy metals is suspected.     

Enzyme-immunoassay for the determination of metallothionein in human urine: application to environmental monitoring

The objectives of this study were to develop an enzyme immunoassay for metallothioneins in human urine using a polyclonal antiserum and to demonstrate a possible relationship between the level of this biomarker and heavy metal exposure. The antiserum was raised in sheep against horse metallothionein conjugated to carboxylated bovine serum albumin. The antibody was used to construct a two-step competitive ELISA procedure. Human urine was treated with activated charcoal powder to remove traces of metallothioneins and known amounts of pure metallothioneins were added to provide standards for a standard curve. Metallothionein levels were measured in two groups of children living in areas of mild and high environmental pollution due mainly to heavy metals. A comparison was made between the biomarker levels and the levels of cadmium and lead in urine samples in the two groups. A group of children from a non-polluted area acted as controls. The results show that the detected levels of metallothioneins appear to correspond to levels of the two heavy metals studied and that there was an apparent relationship to the environmental exposure. Thus according to results of this study the increase in the metallothionein excretion seems to provide an indication of previous of exposure to metals. The ELISA procedure is sensitive and robust and can be used to screen large numbers of samples and is more rapid than the physical procedures currently used for analysis of these proteins. The assay can therefore be used as an additional tool for screening at-risk populations where either environmental or occupational exposure to divalent heavy metals is suspected.     

Enzyme-immunoassay for the determination of metallothionein in human urine: application to environmental monitoring.

The objectives of this study were to develop an enzyme immunoassay for metallothioneins in human urine using a polyclonal antiserum and to demonstrate a possible relationship between the level of this biomarker and heavy metal exposure. The antiserum was raised in sheep against horse metallothionein conjugated to carboxylated bovine serum albumin. The antibody was used to construct a two-step competitive ELISA procedure. Human urine was treated with activated charcoal powder to remove traces of metallothioneins and known amounts of pure metallothioneins were added to provide standards for a standard curve. Metallothionein levels were measured in two groups of children living in areas of mild and high environmental pollution due mainly to heavy metals. A comparison was made between the biomarker levels and the levels of cadmium and lead in urine samples in the two groups. A group of children from a non-polluted area acted as controls. The results show that the detected levels of metallothioneins appear to correspond to levels of the two heavy metals studied and that there was an apparent relationship to the environmental exposure. Thus according to results of this study the increase in the metallothionein excretion seems to provide an indication of previous of exposure to metals. The ELISA procedure is sensitive and robust and can be used to screen large numbers of samples and is more rapid than the physical procedures currently used for analysis of these proteins. The assay can therefore be used as an additional tool for screening at-risk populations where either environmental or occupational exposure to divalent heavy metals is suspected.     

Seroprevalence of Toxoplasma gondii infection and risk factors associated with seropositivity of pregnant women in Korea

A total of 351 serum samples was examined for anti-T. gondii antibody titers; the overall seroprevalence was 3.7%. The Sabin-Feldman dye test (DT), latex agglutination test (LAT), and IgG enzyme-linked immunosorbent assay (IgG ELISA) indicated seroprevalences of 3.7%, 3.4%, and 4.0%, respectively. Compared with the DT, the sensitivities of the LAT and IgG ELISA were 84.6% and 100.0%, respectively, and the specificities of the LAT and IgG ELISA were both 99.7%. An increase in T. gondii seroprevalence with increasing age was detected, but the difference was not significant. The overall seroprevalence of T. gondii-antibody titers in pregnant Korean women was relatively low compared to those of Europeans and Americans. A multivariate analysis of risk factors showed that T. gondii infection was positively correlated with eating raw meat, but was not associated with the consumption of unwashed vegetables, drinking untreated water, a history of raising a cat, or blood transfusion. The consumption of raw or undercooked meat may, therefore, be the main route of T. gondii infection in Korea.     

Fluorometric assay of DNA in cartilage explants using Hoechst 33258

A simple two-step fluorometric assay of DNA in cartilage explants, utilizing the bisbenzimidazole dye Hoechst 33258, is described. Cartilage explants were prepared for assay by digestion with papain. Aliquots of the digest were mixed with dye solution, and the fluorescence emission measured. The enhancement in fluorescence of dye was specific for DNA, as demonstrated by 97% sensitivity to DNase and resistance to RNase. In addition, little or no interference was caused by non-DNA tissue components, since DNA caused an equal enhancement in fluorescence independent of the presence of papain-digested cartilage. By performing the assay on isolated chondrocytes, the cellular content of DNA was computed to be 7.7 pg per chondrocyte. The assay was stable for at least 2 h and sensitive to as little as 6 ng of DNA or equivalently less than 1000 cells. This procedure offers advantages over other established DNA assays of cartilage and may be especially useful in metabolic studies of cartilage explants.     

Purity of commercial non-certified European samples of Pyronin Y

Summary The purity of six European non-certified samples of Pyronin Y was compared with that of two American samples certified by the Biological Stain Commission. The methods used were spectrophotometry and a Methyl Green-Pyronin staining test (both as applied by the Biological Stain Commission), thin layer chromatography, mass spectrometry, determination of pH, and content of some electrolytes. It was found that none of the European batches of Pyronin Y passed the complete test as prescribed by the Biological Stain Commission. Their dye content was uniformly low (between 5 and 19%). Furthermore, thin layer chromatography and mass spectrometry revealed that two of the dye samples contained no Pyronin Y or only traces.It is concluded that assessment of an unknown sample of a dye labelled Pyronin Y should be initiated with thin layer chromatography. The pH and content of electrolytes in an aqueous solution of the dye should also be determined in order to obtain reproducible staining results. Finally, the value of the work performed by the Biological Stain Commission is underlined, although more sophisticated methods are necessary for testing the purity of dyestuffs.     

Detection of autoantibodies to the diabetes-associated antigen IA-2 by a sensitive enzyme-linked immunosorbent assay.

The tyrosine phosphatase like protein IA-2 is an important autoantigen in insulin-dependent diabetes mellitus (type 1 diabetes). Autoantibodies to IA-2 (IA-2A) are present in the serum of patients with type 1 diabetes even before the onset of the disease. Previously, we reported on a radioimmune assay to detect IA-2A, using E. coli-derived 125I-labelled IA-2 as antigen. Although this assay could be shown to be equivalent to the common reference method for IA-2A detection (radioligand assays using in vitro synthesised 35S-methionine labelled antigen), the disadvantages of both assays with respect to synthesis and handling of the radioactive antigen limit their use in routine laboratories. In this study, we have evaluated a non-radioactive enzyme-linked immunosorbent assay (ELISA) for the simple detection of IA-2A. We report on an ELISA where the biotinylated intracytoplasmic part of IA-2 (IA-2ic) is captured on streptavidin-coated plates. The sensitivity of the ELISA was similar to the validated radioligand assay, as it detected 47 of 69 (69%) patients with type 1 diabetes as compared to 46 of 68 (67 %) with the reference method for IA-2A detection (radioligand assays using in vitro synthesised 35S-methionine labelled antigen). Only 2 of 50 (4%) patients with autoimmune thyroid disease and 1 of 114 (1 %) healthy controls were detected in the ELISA, confirming specificity. There was a significant correlation between the ELISA and the radioligand assay (r = 0.64, p<0.001). We conclude that this ELISA is suitable to detect IA-2A in the serum of patients with type 1 diabetes with a similar sensitivity and specificity to the radioligand assay. This ELISA will allow rapid and simple measurement of IA-2A where the radioligand assay is inconvenient or not available.     

A novel method for measuring human lipoprotein lipase and hepatic lipase activities in postheparin plasma

The objective of this study was to establish a new lipoprotein lipase (LPL) and hepatic lipase (HL) activity assay method. Seventy normal volunteers were recruited. Lipase activities were assayed by measuring the increase in absorbance at 546 nm due to the quinoneine dye. Reaction mixture-1 (R-1) contained dioleoylglycerol solubilized with lauryldimethylaminobetaine, monoacylglycerol-specific lipase, glycerolkinase, glycerol-3-phosphate oxidase, peroxidase, ascorbic acid oxidase, and apolipoprotein C-II (apoC-II). R-2 contained Tris-HCl (pH 8.7) and 4-aminoantipyrine. Automated assay of lipase activities was performed with an automatic clinical analyzer. In the assay for HL + LPL activity, 160 microl R-1 was incubated at 37 degrees C with 2 microl of sample for 5 min, and 80 microl R-2 was added. HL activities were measured under the same conditions without apoC-II. HL and LPL activities were also measured by the conventional isotope method and for HL mass by ELISA. Lipase activity detected in a 1.6 M NaCl-eluted fraction from a heparin-Sepharose column was enhanced by adding purified apoC-II in a dose-dependent manner, whereas that eluted by 0.8 M NaCl was not. Postheparin plasma-LPL and HL activities measured in the present automated method had high correlations with those measured by conventional activity and mass methods. This automated assay method for LPL and HL activities is simple and reliable and can be applied to an automatic clinical analyzer.     

Novel fluorescent trimethine cyanine dye 7519 for amyloid fibril inhibition assay

Abstract

Fluorescence spectroscopy was used to study the ability of dye 7519 to follow the transition of monomeric insulin into fibrils and applicability of the dye to the insulin aggregation inhibition assay. The commercially available classic amyloid stain, thioflavin T, was used as the reference dye. For selecting potential inhibitors, the QSAR approach was applied. Dye 7519 appeared to be suitable for monitoring insulin aggregation into fibrils in vitro. The properties of the dye allowed us to test it as a potential probe in the screening assay of potential inhibitors of insulin fibrillization. One hundred forty-four flavonoids were tested as potential inhibitors of amyloid fibril formation using the quantitative structure activity relationship approach. Among them, 10 candidates with high indexes of inhibition were selected for tests in vitro using dye 7519 and the reference amyloid dye thioflavine T. Using dye 7519 fluorescence, we found that two compounds had inhibitory effects on insulin amyloid formation. These results agree with inhibition data using the thioflavine T assay. Our studies demonstrated that the fluorescent cyanine dye 7519 is a sensitive probe for quantitative detection of insulin amyloid formation and can be applied to screen agents capable of affecting aggregation of amyloid proteins.     

Microplate screening for apoptosis with antibody to single-stranded DNA distinguishes anticancer drugs from toxic chemicals

The effect of anticancer drugs and toxic compounds on cultures of human leukemic cells was evaluated by an enzyme-linked immunosorbent assay (Apoptosis ELISA) that uses a monoclonal antibody against single-stranded DNA to quantitate the apoptotic cells. The concentrations of 13 anticancer drugs, which increased Apoptosis ELISA absorbance, were close to the cytotoxic concentrations determined by the long-term cell survival assay. Short-term tetrazolium-based microculture tetrazolium (MTT) assay was significantly less sensitive than the Apoptosis ELISA and the cell survival assay for all anticancer drugs. For 6 drugs, cytotoxic concentrations measured by the MTT assay were at least 1 log higher than the concentrations inducing apoptosis. Importantly, in contrast to the anticancer drugs, 14 toxic chemicals did not increase the Apoptosis ELISA absorbance at cytotoxic concentrations. The difference in apoptosis induction by the anticancer drugs and the toxic chemicals was especially large in cultures treated with drug concentrations 2-fold higher than the IC(50) dose. Although all of the anticancer drugs tested induced intense ELISA reaction (mean absorbance 2.0), all toxic chemicals tested did not induce apoptosis. The Apoptosis ELISA assay could have useful applications in drug development as it can distinguish between clinically useful anticancer drugs and toxic compounds, has sensitivity similar to that of the long-term cell survival assay, and provides insight into the mechanism of drug cytotoxicity by differentiating between compounds killing cells by apoptosis and necrosis.     

Use of murine myeloma protein M467 for detecting Salmonella spp. in milk.

This investigation introduces the use of an immunoglobulin A mouse myeloma protein for the detection of Salmonella spp. in milk. The immunoglobulin A protein M467 reacts with flagellin from a wide variety of serotypes. Two assays were developed which used an enzyme-linked immunosorbent assay (ELISA) and M467. Alkaline phosphatase was conjugated to M467 (M467-PH), and the presence of Salmonella dublin was detected by a competitive solid-phase ELISA and a membrane filtration ELISA. The competitive assay competed viable Salmonella spp. found in contaminated milk against polymerized flagellin or whole bacteria fixed to polyvinyl plates for binding by M467-PH. The membrane filtration method utilized a hydrophilic membrane for filtering the bacteria, which were then detected by the reaction with M467-PH and substrate. The sensitivity of the competitive solid-phase ELISA was 10(3) bacteria ml-1, whereas the filter membrane assay required the media containing the bacteria to be cultured in enrichment medium for 4 h before the assay to ensure detection. Either assay could be run within a typical 8-h work day. The filter membrane assay was not suitable for milk due to the high level of natural alkaline phosphatase activity in the liquid food.