Direct Labeling Technique Research Articles

Direct TAMRA-dUTP labeling of M. tuberculosis genes using loop-mediated isothermal amplification (LAMP)

Fluorescent molecule-based direct labeling of amplified DNA is a sensitive method employed across diverse DNA detection and diagnostics systems. However, using pre-labeled primers only allows for the attachment of a single fluorophore to each DNA strand and any modifications of the system are less flexible, requiring new sets of primers. As an alternative, direct labeling of amplified products with modified nucleotides is available, but still poorly characterized. To address these limitations, we sought a direct and adaptable approach to label amplicons produced through Loop-mediated isothermal amplification (LAMP), using labeled nucleotides (dUTPs) rather than primers. The focus of this study was the development and examination of a direct labeling technique of specific genes, including those associated with drug resistance in Mycobacterium tuberculosis. We used 5-(3-Aminoallyl)-2′-deoxyuridine-5′triphosphate, tagged with 5/6-TAMRA (TAMRA-dUTP) for labeling LAMP amplicons during the amplification process and characterized amplification and incorporation efficiency. The optimal TAMRA-dUTP concentration was first determined based on amplification efficiency (0.5% to total dNTPs). Higher concentrations of modified nucleotides reduced or completely inhibited the amplification yield. Target size also showed to be determinant to the success of amplification, as longer sequences showed lower amplification rates, thus less TAMRA incorporated amplicons. Finally, we were able to successfully amplify all four M. tuberculosis target genes using LAMP and TAMRA-modified dUTPs.

Designed Synthesis of Dual Emitting Silicon Quantum Dot for Cell Imaging: Direct Labeling of Alpha 2-HS-Glycoprotein.

The innocent silicon quantum dots (SQDs) having dual emissive property (blue in VIS and red in NIR), high photostability, and freedom from auto fluorescence are designed and synthesized for the first time using ethylene glycol. A new attempt has been made for direct labeling of Alpha 2-HS-Glycoprotein (Fetuin A) through functionalization of the synthesized dots by EDC coupling. The SQDs were characterized by FTIR, TEM, AFM, XRD, EDX, DLS, and TGA. The chemistry involved in the synthesis and functionalization of dots is elucidated in detail. The synthesized SQDs are suitable for live cell imaging as well as direct labeling of the Fetuin A in the NIR region. The direct labeling technique developed for Fetuin A imaging is robust, more specific, and simple, and reduces the number of incubation and washing steps and produces better quality data compared to the conventional method using Rhodamine B.

Preparation and biological evaluation of 99mTc-TMPP as a novel agent for tumor diagnosis

Compound 5, 10, 15, 20 tetrakis {4-methoxyphenyl} 21H, 23H porphyrin (TMPP) was labeled with technetium-99m via direct labeling technique using stannous chloride as a reducing agent. The optimum conditions that gave high labeling yield (95.2%) of 99mTc-TMPP complex were achieved by using 3mg TMPP, 100μg SnCl2.2H2O, at pH 3 and 30min reaction time. Molecular geometry was used to illustrate the structure of complex which is formed between 99mTc and TMPP. The preclinical evaluation and biodistribution in solid tumor bearing mice showed high biological accumulation in solid tumor cells (22.66 % injected activity/ g tissue) and high T/NT ratio equal to 3.21 ± 0.15 at 30 minutes post-injection. Data described before could recommend 99mTc-TMPP as a potential targeting radiopharmaceutical for tumor imaging.

Preparation of (99m)Tc-labelled methotraxate by a direct labeling technique as a potential diagnostic agent for breast cancer and preliminary clinical results.

Methotrexate (MTX) is being used in clinical oncology for the treatment of a wide variety of cancers. The aim of the present study was to label directly MTX with (99m)Tc by using Sn/pyrophosphate as reducing agent and to use this labeled compound as a potential anticancer radiopharmaceutical for breast cancer imaging. We studied the labeling efficiency of the (99m)Tc-MTX compound by paper chromatography and instant thin layer chromatography (ITLC) in acetone and saline and found it to be more than 95%. In vitro stability of labeled MTX in serum was studied up to 5h. Partition coefficient in n-octanol and saline indicated that the labeled radiopharmaceutical was hydrophilic. We then tested (99m)Tc-MTX in 5 breast cancer female patients. Protein bound (99m)Tc-MTX showed rapid clearance from blood. The biodistibution data suggested that (99m)Tc-MTX was cleared by the kidneys and the liver. Patients ' data also showed highly significant uptake of (99m)Tc-MTX in breast cancer. In conclusion, this study indicated that (99m)Tc-MTX may be used as a potential diagnostic agent for breast cancer patients imaging and may show treatment efficiency in case MTX is to be used for treatment.

Stem Cells as a Tool for Breast Imaging

Stem cells are a scientific field of interest due to their therapeutic potential. There are different groups, depending on the differentiation state. We can find lonely stem cells, but generally they distribute in niches. Stem cells don't survive forever. They are affected for senescence. Cancer stem cells are best defined functionally, as a subpopulation of tumor cells that can enrich for tumorigenic property and can regenerate heterogeneity of the original tumor. Circulating tumor cells are cells that have detached from a primary tumor and circulate in the bloodstream. They may constitute seeds for subsequent growth of additional tumors (metastasis) in different tissues. Advances in molecular imaging have allowed a deeper understanding of the in vivo behavior of stem cells and have proven to be indispensable in preclinical and clinical studies. One of the first imaging modalities for monitoring pluripotent stem cells in vivo, magnetic resonance imaging (MRI) offers high spatial and temporal resolution to obtain detailed morphological and functional information. Advantages of radioscintigraphic techniques include their picomolar sensitivity, good tissue penetration, and translation to clinical applications. Radionuclide imaging is the sole direct labeling technique used thus far in human studies, involving both autologous bone marrow derived and peripheral stem cells.

BIODISTRIBLTION OF LABELED IOD0-AZATHIOPRINE IN EAC TUMOR BEARING MICE

Azathioprine (Aza) is an antimetabolite drug, could be labeled with the Auger emitters todine-125 Aza could be used as an ideal vehicle to deliver radioactive decay energy to DNA of tumor cells causing DNA double strand break, thus stop DNA synthesis. In this study, the process of labeling was done via direct labeling technique using chloramine -T as an oxidizing agent and heating to 75 °C, for 30 minutes at pH 7 using 0.5 M phosphate buffer .The radiochemical purity of the labeled compound at the above Conditions, was determined using electrophoresis technique and was above 90% About 2.5 x 106 of Ehrlich Ascites Carcinoma (EAC ) was Injected intrapritoneally (i.p) to produce stiles and intramuscularly (i.m) in the right thigh to produce solid tumor in female mice. Biodistribution studies were carried out by injecting solution of 125I-Aza in normal and tumor bearing mice The uptake in ascites was over 40 % of the injected dose at 12h post injection and above 20% in solid tumor. The data revealed localization of the tracer in the tumor tissues with high percentage sufficient to give radiotherapeutic effect as well as promising tool for diagnosis.

Labeling peptides with rhenium-188

A direct labeling technique via EHDP for the preparation of 188Re-somatostatin analogue peptide β-(2-naphthyl)-D-Ala–Cys–Tyr-D-Trp–Lys–Val–Cys–Thr–amide complex was developed. The influence of reaction conditions such as pH, temperature, weak ligand concentration and stannous chloride concentration were investigated. Methods of analysis were also established permitting identification of radiochemical impurities which may be present in the radiopharmaceutical solution. Results showed that under the procedure reported herein 188Re–peptide complex can be prepared with a radiochemical purity of 90% and a specific activity up to 1.8 GBq mg −1 without radiolytic degradation of the product.

Qualitative and Quantitative Assessment of Modified In Situ Reverse Transcription-Polymerase Chain Reaction (In Situ RT-PCR) Method.

The in situ reverse transcription-polymerase chain reaction (in situ RT-PCR) method has been used to detect low levels of expression of mRNA in cells and tissues. We applied a newly developed in situ RT-PCR to cells and quantitatively compared the sensitivity of the in situ RT-PCR with that of conventional in situ hybridization (ISH). Human tumor cell lines with either high, moderate, low or no expression of IL-6 mRNA as determined by in vitro competitive RT-PCR were used. In situ RT-PCR was performed using Thermus thermophilus DNA polymerase, special primer sets to form high molecular weight concatemers and an in situ digoxigenin (DIG) direct labeling technique. Conventional ISH was performed using DIG-labeled cRNA probe. Specific signals of IL-6 mRNA were observed in the cytoplasm of positive cells by both in situ RT-PCR and ISH. In situ RT-PCR was more than 100-fold more sensitive than ISH. The specificity of in situ RT-PCR was confirmed by appropriate control tests. The data indicated that the sensitivity of our in situ RT-PCR applied to culture cells was significantly higher than that of ISH, and that the technique retained high specificity.

Lymphocyte immunoregulatory cells present in semen from human immunodeficiency virus (HIV)-infected individuals: a report from the HIV Heterosexual Transmission Study.

The purpose of this study was to determine the types and distribution of immune subsets present in semen from human immunodeficiency virus (HIV)-infected (HIV+) individuals and to compare these values with those measures in semen from HIV-negative (HIV-) individuals. To accomplish this, a direct three-color monoclonal antibody labeling technique was employed to identify immune cells in fresh ejaculates. Once labeled, the percent of each immune subset present in the ejaculate was determined by flow cytometric analysis. The percent of CD3+ cells present in the semen of the HIV+ group showed no significant difference when compared with semen from the HIV- group. Analysis of the CD4+ subset yielded a significantly lower percent in the HIV+ group than in the HIV- group. The analysis of the CD8+ subset yielded a higher percent of cells present in semen from HIV+ individuals. The CD8 higher value along with lower CD4 value results in a lower CD4/CD8 ratio in the HIV+ group. Further subset studies showed that the percent of cells expressing naive (CD4+ CD45RA+) and memory (CD4+ CD45RO+) markers was lower in the HIV+ group. This study provides additional data supporting the utility of flow cytometry and monoclonal antibodies to immunophenotypic cells present in semen ejaculates. It is also the first reported application of the technique to a disease-based model and may be useful to better understand issues of mucosal immunity and transmission of sexually transmitted diseases such as HIV.

Direct immunofluorescent labeling of cells.

In their original immunofluorescent labeling technique, Coons and colleagues employed a one-step, direct labeling of antigen using a fluorescein-conjugated antibody (, ). Although this was not the first published report of a chemically modified antibody, it was the first time the antibody was used as a tool to examine an antigen in situ. Shortly thereafter, this method was adapted, giving rise to the indirect labeling technique (). Rather than conjugating a marker molecule to the primary antibody, in this technique, a secondary antibody raised against the γ globulin of the primary species is conjugated with the marker molecule (Fig. 1). Although the direct labeling technique has the advantage of being quick, requiring a single incubation with the labeled reagent and one subsequent wash step, there are disadvantages. Each different primary antibody must be fluorescently labeled, and the resulting fluorescence is weak since only one labeled primary antibody binds to each antigen. The indirect labeling technique adds one more incubation and wash step, but it has the advantage of amplifying the fluorescent signal because several fluorescently labeled secondary antibodies can bind to each primary antibody. In addition, labeled secondary reagents specific for various species’ immunoglobulin classes are readily available commercially, stable in the lyophilized or frozen state, and relatively inexpensive.

Postembedding labeling methods.

Since it was first introduced, postembedding immunogold labeling has become the most widely used method of immunolabeling for electron microscopy. For postembedding labeling, samples are first fixed, embedded, and sectioned. All immunostaining is performed on sections mounted on grids. The immunostaining may be done with a direct labeling technique with the primary antibody conjugated to colloidal gold, or by an indirect method where the primary antibody is unlabeled and the gold is conjugated to a secondary or tertiary antibody, protein A, protein G, etc. Colloidal gold-antibody conjugates are also widely used, especially in indirect immunocytochemical methods. This chapter gives the standard method for post embedding labeling as well as one in which the cell membranes are enhanced.

The labeling of proteins and LDL with 99mTc: a new direct method employing KBH 4 and stannous chloride

A new direct labeling technique for proteins with technetium-99m ( 99mTc) has been developed and makes use of borohydride and stannous chloride. The method is simple and reproducible and gives a high labeling efficiency and high retention of biological activity for proteins, including polyclonal immunoglobulin (Ig), antifibrin monoclonal antibody, tissue type plasminogen activator, fibrinogen and low density lipoprotein (LDL). This method can be used in kit-format and takes about 20 min preparation time at room temperature. Both in vitro and in vivo studies indicate good stability of the label. In vivo, mice and rabbit images show significant accumulation of 99mTc-Ig at an inflammation area, 99mTc-antifibrin at a thrombus site and 99mTc-LDL in atherosclerotic lesions. The method is attractive for routine research and clinical purposes.

Labeling and stability of radiolabeled antibody fragments by a direct 99mTc-labeling method

The in vitro labeling and stability of 99mTc-labeled antibody Fab′ fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 ( N = 5), IgG2a ( N = 2) and IgG3 ( N = 1) isotypes were labeled with a preformed 99mTc- d-glucarate complex. No loss of radioactivity incorporation was observed for all the 99mTc-labeled antibody fragments after 24 h incubation at 37 °C. The 99mTc-labeled antibody fragments (IgG1, N = 2; IgG2a, N = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Furthermore, using the affinity chromatography technique, two of the 99mTc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 °C. The bonding between 99mTc and antibody fragments was elucidated by challenging with a diamide ditholate (N 2S 2) compound. The Fab′ with IgG2a isotype displayed tighter binding to 99mTc in comparison to the Fab′ from IgG1 and IgG3 isotype in N 2S 2 challenge and incubation with human plasma. The in vivo biodistribution of five 99mTc-labeled fragments were evaluated in normal mice. In conclusion, the direct labeling method allows stable 99mTc labeling of antibody fragments from three of the major murine isotypes.

Further evidence for entry of infectious bovine rhinotracheitis virus into bovine kidney cells

The penetration of bovine kidney cells by infectious bovine rhinotracheitis virus, a member of the herpesvirus group, was investigated using the direct immunoferritin labeling technique. Electron microscopic examination of infected cells after 10 min at 37°C revealed fusion between viral envelope and cell membrane; the former reacted with the ferritin particles conjugated with antiviral antibody. However, shortly after penetration of the nucleocapsid, viral-specific antigenic sites on the plasma membrane were not detected by the immunoferritin technique. Antigenically reactive structures in a disorganized array were frequently detected extracellularly, situated above the penetration sites as indicated by the internalized nucleocapsids.