Development of a new magnetic beads-based immunoprecipitation strategy for proteomics analysis

Sample pre-treatment is a critical step for an efficient and reliable analysis and it is highly dependent on the complexity of the matrix. This work shows an example of application of an immunoprecipitation approach using a new magnetic beads-based format, which allows a selective/specific extraction of potential biomarkers from metastatic prostate cancer. Results obtained on the development of this method, and its application for the extraction and pre-concentration of certain biomarkers present in metastatic cell lines of prostate cancer, are presented and discussed. It is concluded that the efficiency of the immunoprecipitation step is clearly compromised by the crosslinking conditions and it is highly dependent on the specificity of selected antibodies. The epoxy magnetic beads used in this work allowed an effective crosslinking of the antibodies contributing to an increased efficiency of the immunoprecipitation step. The optimized conditions for the application of these epoxy magnetic beads for the immunoprecipitation of anti-TUBA3C in metastatic prostate cancer cell line (PC3) are discussed here, as an example of application of the immnuprecipitation approach developed, which resulted in a very efficient tool for a specific extraction and pre-concentration of the targeted protein and, therefore, contributing to the efficiency of further analysis.     

Effects of ethanol alone or after pretreatment with 20% ethanol on phospholipid metabolism in rat gastric mucosa

Changes in phospholipid metabolism in gastric mucosa caused by instillation of absolute ethanol (a cell-damaging agent) into the stomach of rats and the effects of pretreatment with 20% ethanol (a mild irritant) were investigated by using radioisotope-labeled fatty acids and glycerol. The labeled precursors were incorporated mainly into phosphatidylcholine and triacylglycerol, and also to lesser extents into phosphatidylethanolamine and phosphatidylinositol + phosphatidylserine. The instillation of absolute ethanol reduced the incorporation of fatty acids and glycerol into phospholipids within 15 min, indicating the inhibition by ethanol of de novo synthesis of phospholipids. Pretreatment with 20% ethanol caused the incorporation of fatty acids into phospholipids to be maintained after absolute ethanol instillation. These results suggest that the pretreatment with 20% ethanol may protect the cellular synthetic activity of phospholipids against damage by absolute ethanol. The incorporation of fatty acids into the free fatty acid fraction, monoacylglycerol and diacylglycerol was increased by absolute ethanol instillation, suggesting damage to the blood vessels of the gastric mucosa, and these changes were inhibited to some extent by the pretreatment with 20% ethanol.     

Enzyme-linked immunosorbent assay for determination of cyclosporin a in the whole blood

A test-system based on enzyme-linked immunosorbent assay (ELISA) for quantitative determination of cyclosporin A (CSA) in human whole blood has been developed. The detection limit of the method was 25 ng/ml, the linearity of the method in the concentration range of 60–1400 ng/ml varied from 94 to 105%, the variation coefficient did not exceed 8%. The novel method exhibited good correlation with radioimmunoassay and polarization fluoroimmunoassay methods; the linear regression coefficients were 0.965 and 0.984, respectively. The developed test system is stable for at least 9 months when stored at 4°C and can be used in clinical practice.     

Radiosensitization of murine tumors by Fluosol-DA 20%

The effect of perfluorochemicals in combination with carbogen breathing on the response of SCK tumors of mice to fractionated irradiation was investigated. The SCK tumors of A/J mice were irradiated twice a day at 3 Gy per fraction (6 Gy per day), with a total dose of 18 Gy over 3 days. When the host animals were treated with an intravenous (iv) injection of 12 ml/kg of Fluosol-DA 20% before the first daily tumor irradiation and carbogen breathing during every X irradiation with Fluosol-DA 20% injection without carbogen breathing. The hypoxic cell fraction, as determined by an in vivo-in vitro cloning assay, decreased significantly, and the intratumor pO2, as determined with microelectrodes, was markedly increased by Fluosol-DA 20% injection and carbogen breathing. It was concluded that oxygenation of hypoxic cells in SCK tumors during the course of fractionated irradiation was improved by the iv injection of Fluosol-DA 20% and carbogen breathing.     

A faster immunofluorescence assay for tracking infection progress of human cytomegalovirus

Immunofluorescence assay (IFA) is one of the most frequently used methods in the biological sciences and clinic diagnosis, but it is expensive and time-consuming. To overcome these limitations, we developed a faster and more cost-effective IFA (f-IFA) by modifying the standard IFA, and applied this method to track the progression of human cytomegalovirus (HCMV) infection in different cells. The f-IFA that we developed not only saves time, but also dramatically reduces the quantity of antibody (Ab), which will facilitate the application of IFA in clinic diagnosis. f-IFA requires only 15 min for blocking, 10 min incubation for each primary and secondary Abs, followed by 1 min extensive wash after each incubation. Only 25 μl of diluted Ab solution was needed for each coverslip at the primary and secondary Ab incubation steps. In addition, all steps were performed at room temperature. This f-IFA has been applied successfully to follow virion entry (pp65) and expression of viral genes (IE1, UL44, and pp65) in order to track the details of HCMV infection process. We found that ～0.5% HCMV-infected T98G cells formed multiple-micronuclei (IE1 and nucleus staining) and had virus shedding (pp65 staining) by f-IFA, which could not be detected by the traditional IFA. Our results indicated that f-IFA is a sensitive, convenient, fast, and cost-effective method for investigating the details of virus infection progress, especially HCMV infection. The faster and cost-effective feature with higher sensitivity and specificity implies that f-IFA has potential applications in clinical diagnosis.     

Determination of alginate composition by a simple enzymatic assay

The action of alginate lyases may be easily followed in a UV-spectrophotometer, since each cut of the alginate chain will create an unsaturated unit at the non-reducing end with a strong absorbance at 230 nm. During prolonged incubation, this absorbance will approach an apparent endpoint level that reflects the initial substrate concentration. On this basis, a standardized assay has been developed. A combination of purified mannuronate lyase from Haliotis tuberculata and purified guluronate lyase from Klebsiella pneumoniae is applied to get quantitative concentration estimates that do not depend on alginate composition. The production of alginate in Azotobacter vinelandii is included as an example of application. Most important, by applying both enzymes alone and in combination, the block composition of the alginate may be estimated. Data for a series of widely different alginates have been compared with those obtained by NMR.     

Serodiagnosis of cilia-associated respiratory bacillus infection by the indirect immunofluorescence assay technique

Antibody to cilia-associated respiratory (CAR) bacillus was detected by the indirect immunofluorescence assay (IFA) technique using tracheal sections of infected mice as antigen in serum samples collected from rats infected naturally and experimentally. Nine of 23 cases of natural infection were positive in IFA antibody, with titres ranging from 1:10 to 1:80, and all these antibody-positive cases were also histologically positive. The remaining 14 cases were negative in both IFA antibody and histological diagnosis, even though some of them were infected with Sendai virus and Mycoplasma pulmonis. In the experimental infection, serum samples collected from 18 rats on days 4, 7, 14, 21, 28 and 56 post-inoculation (PI) (three rats for each point) and examined for IFA antibody revealed that seroconversion occurred in one rat on day 14 PI and in three rats on day 21 PI. Antibody titres of 1:80 to 1:160 remained to the termination of the experiment. The IFA technique was useful for the diagnosis of CAR bacillus infection except in the early stage of the infection.     

Reactivity of serum samples from patients with a flavivirus infection measured by immunofluorescence assay and ELISA

Flavivirus infections are a significant public health problem, since several members of the Flaviviridae family are highly pathogenic to humans. Accurate diagnosis and differentiation of the infecting virus is important, especially in areas where many flaviviruses are circulating. In this study we evaluated a newly developed commercially available immunofluorescence assay (IFA) (INDX, Baltimore, MD, USA) for the detection of IgM and IgG antibodies against dengue virus, yellow fever virus, Japanese encephalitis virus and West Nile virus. IFA was compared with standard diagnostic enzyme immunoassays (EIAs) specific for the detection of IgM and IgG antibodies against these viruses. Forty-seven serum samples from patients with a defined flavivirus infection were tested. As controls, serum samples from individuals with antibodies against tick-borne encephalitis virus and hepatitis C virus as well as healthy individuals were included. The results obtained from this study indicate that IFA showed a significantly better discrimination for flavivirus specific IgM antibodies than did the standard IgM specific EIAs (the overall cross-reactivity varied between 4 and 10% by IFA and 30-44% by EIA for the respective viruses). In contrast, the detection of flavivirus specific IgG antibodies showed high cross-reactions in both IFA and EIAs (overall cross-reactivity 16-71 and 62-84%, respectively). This study clearly stated the complexity of flavivirus diagnosis, showing that one cannot rely on one assay or search for one virus only. The flavivirus IFA is a useful tool for the identification of flavivirus infections during the acute stage of disease. In particular, IFA can be an important diagnostic tool for testing samples from travellers who have been accidentally exposed to these viruses.     

A comparison of kinematics between overarm throwing with 20% underweight, regular, and 20% overweight balls

The aim of this study was to compare the kinematics in throwing with a regular weighted handball with 20% lighter and heavier balls in female experienced handball players. In total, eight joint movements during the throw were analyzed. The analysis consisted of maximal angles, angles at ball release, and maximal angular velocities of the joint movements and their timings during the throw. Results on 24 experienced female team handball players (mean age 18.2 ± 2.1 years) showed that the difference in ball weight affected the maximal ball velocity. The difference in ball release velocity was probably a result of the significant differences in kinematics of the major contributors to overarm throwing: elbow extension and internal rotation of the shoulder. These were altered when changing the ball weight, which resulted in differences in ball release velocity.     

Determination of the DNA-synthesizing cell count by the immunofluorescence method

In DNA-synthesizing cells DNA is partially single-stranded. Anti-thymidine antibodies, while specifically reacting with this DNA, form a complex which may be revealed using indirect immunofluorescent technique. A comparative determination of DNA-synthesizing cell number in tumor tissue (larynx squamous cell carcinoma) was performed using immunofluorescent technique and radioautography. The former method showed the labeling index (LI) to vary from 1.2 to 9.9%, while the latter showed it to vary from 1.0 to 8.2%. The correlation ratio between the LI values obtained by the two techniques was 0.79. To eliminate a possible reaction of anti-thymidine antibodies with cellular RNA, specimens were preincubated in solutions with RNAase. No more than 6 hours were required to stain specimens using this LI estimation technique. This investigation allows to reveal DNA synthesizing cells not only in the periphery of a histological section, as does routinely radioautography, but also in its centre.     

Preservation of immunorecognition by transferring cells from 10% neutral buffered formalin to 70% ethanol

Abstract

Prolonged fixation of cells and tissues in 10% neutral buffered formalin (NBF) may decrease immunorecognition in some antigen-antibody pairs. Short fixation in 10% NBF followed by transfer to 70% ethanol has been used to overcome these effects, but the effects of this transfer on immunorecognition have not been explored adequately. We used two cell lines, DU145 (prostate cancer) and SKOV3 (ovarian cancer), grew them on coverslips and fixed them with 10% NBF at room temperature for 5 min and 12, 15, 18, 36, 108 and 180 h. Aliquots of the same cells were fixed in 10% NBF for 12 h, then transferred to 70% ethanol for 3, 6, 24, 96 and 168 h. Immunostaining with PCNA, Ki67-MIB-1, cytokeratins AE1/AE3 and EGFr was done concomitantly. In both cell lines, immunorecognition decreased between 18 and 36 h of fixation in 10% NBF for PCNA, Ki67-MIB-1 and cytokeratins AE1/AE3. By 108 to 180 h of 10% NBF exposure, there was complete loss of immunorecognition of PCNA and extensive loss of Ki67-MIB-1 and cytokeratins AE1/AE3. The effects on EGFr immunorecognition were less. Transfer to 70% ethanol after fixation for 12 h in 10% NBF preserved immunorecognition of the antibodies.     

Determination of phospholipid hydroperoxides in human blood plasma by a chemiluminescence-HPLC assay

A chemiluminescence-high performance liquid chromatography (CL-HPLC) system was developed (Miyazawa, T. et al., Anal. Lett., 20, 915-925, 1987) and applied for the hydroperoxide-specific determination of phosphatidylcholine hydroperoxide (PCOOH) in biological tissues such as human blood plasma (Miyazawa, T. et al., Anal Lett 21:1033-1044, 1988; J. Biochem. 103:744-746; 1988). This system involves separation of phosphatidylcholines from plasma total lipids with normal phase silica gel HPLC and post-column detection of hydroperoxide-dependent chemiluminescence of PCOOH. The chemiluminescence is produced by luminol oxidation during a reaction of hydroperoxide and cytochrome c-heme. The high specificity for hydroperoxide base enables a sensitive assay for a large range of PCOOH, with the detection limit of 10 picomole of hydroperoxide-O2. By use of this assay system, the presence of PCOOH in human blood plasma is confirmed quantitatively. The PCOOH concentration of healthy plasma is in the range below 10 nM to 500 nM, and much higher concentrations (500-9000 nM) of PCOOH are observed in the plasma of unhealthy donors.     

Comparison of an enzyme-linked immunosorbent assay, an immunofluorescence assay and a hemagglutination inhibition assay for detection of antibodies to K-papovavirus in mice

The sensitivity of a newly developed enzyme-linked immunosorbent assay (ELISA) for detection of antibody to K virus was compared with the sensitivities of an immunofluorescence assay (IFA) and a hemagglutination inhibition assay (HIA). Specific pathogen-free BALB/c RIVM mice, 5 weeks old, were inoculated intraperitoneally with a mouse organ suspension containing 10(4.5) TCID50 of K virus per dose. Control animals were inoculated with a control mouse organ suspension. No clinical signs were observed during the 7 weeks they were followed for the development of serum antibody. The ELISA proved to be the most sensitive of the three assays and demonstrated K virus-specific antibodies as early as 3 days after infection.     

Effect of operant self-administration of 10% ethanol plus 10% sucrose on dopamine and ethanol concentrations in the nucleus accumbens

Although operant ethanol self-administration can increase accumbal dopamine activity, the relationship between dopamine and ethanol levels during consumption remains unclear. We trained Long-Evans rats to self-administer escalating concentrations of ethanol (with 10% sucrose) over 7 days, during which two to four lever presses resulted in 20 min of access to the solution with no further response requirements. Accumbal microdialysis was performed in rats self-administering 10% ethanol (plus 10% sucrose) or 10% sucrose alone. Most ethanol (1.6 +/- 0.2 g/kg) and sucrose intake occurred during the first 10 min of access. Sucrose ingestion did not induce significant changes in dopamine concentrations. Dopamine levels increased within the first 5 min of ethanol availability followed by a return to baseline, whereas brain ethanol levels reached peak concentration more than 40 min later. We found significant correlations between intake and dopamine concentration during the initial 10 min of consumption. Furthermore, ethanol-conditioned rats consuming 10% sucrose showed no effect of ethanol expectation on dopamine activity. The transient rise in dopamine during ethanol ingestion suggests that the dopamine response was not solely due to the pharmacological properties of ethanol. The dopamine response may be related to the stimulus properties of ethanol presentation, which were strongest during consumption.     

Determination of calmodulin by competitive binding assay

Calmodulin levels in tissue or cellular extracts can be determined by competition with 125I-calmodulin in a filtration-based direct binding assay. The method is rapid, uses readily available stable components, and possesses a selectivity and sensitivity comparable to that observed with immunoassay and phosphodiesterase activation. This assay provides a tool to readily probe changes in calmodulin levels in cells and tissues as a function of pathophysiologic state.     

Lipoprotein uptake by vascular smooth muscle cells of the rat cultured in 5% or 20% oxygen

Freshly dispersed aortic smooth muscle cells of the rat were grown in either 5% or 20% oxygen. Cells proliferated more rapidly in 5% than in 20% oxygen although at confluency the protein content per cell was 20% less in the 5% than in the 20% oxygen environment. By electron microscopy, cell morphology was the same in both environments. Uptake of low-density and high-density homologous lipoprotein was unaffected by oxygen tension. Other studies, however, showed that induction of receptors for lipoprotein binding behaved variably in the low compared to the high oxygen condition. These differences were not associated with differences in lipid synthesis in the two conditions.     

Microscale high-performance liquid chromatography–electrospray tandem mass spectrometry assay for cyclosporin A in blood

To facilitate quantitative analysis of cyclosporin A in low volume blood samples we developed a sensitive and specific microscale reversed-phase HPLC–electrospray tandem mass spectrometry assay. Blood samples (100 μl) were prepared by acetonitrile precipitation and C₁₈ solid-phase extraction. Detection was by multiple-reactant monitoring. The method was linear over the range 5–1000 μg/l (r≥0.997) with accuracy between 95.4 and 102.0% over this range. Total imprecision was 11.1% at 10 μg/l and 2.8% at 800 μg/l. Absolute recovery of cyclosporin A and internal standard was 72.5 and 73.3%, respectively. When this method was evaluated against a conventional HPLC with UV detection, in patient samples, they were interchangeable (y=0.988x+10.0, r=0.996). This HPLC–ESI-MS–MS method will be applicable to therapeutic monitoring in paediatric transplant patients and multiple point pharmacokinetic studies in animals and humans.