Optimal Labeling Conditions Research Articles

Time-resolved fluorescence using a europium chelate of 4,7-bis-(chlorosulfopheny1)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA): Labeling procedures and applications in immunoassays

We describe optimal conditions for protein labeling with a new fluorescent probe, 4,7-bis-(chlorosulfophenyl)-1, 10-phenanthroline-2,9-dicarboxylic acid (BCPDA). The labeled proteins are suitable for time-resolved fluorometric applications because BCPDA forms fluorescent comlexes with Eu 3+ which exhibit very long fluorescence lifetimes. Labeling parameters such as the organic t used, pH, protein concentration, BCPDA excess and incubation times, were optimized accordingly. Excess BCPDA was removed by gel filtration and labeled proteins were characterized by absorbance and fluorescence measurements. The effect of labeling on the biological (binding) activity of the proteins streptavidin, avidin, monoclonal and polyclonal antibodies was also studied. It is shown that the labeled antibodies can be used for time-resolved fluoroimmunoassay applications.

An artificial test substrate for evaluating electron microscopic immunocytochemical labeling reactions.

We describe an artificial substrate system for optimization of labeling parameters in electron microscope immunocytochemical studies. The system involves use of blocks of glutaraldehyde-polymerized BSA into which a desired antigen is incorporated by a simple soaking procedure. The resulting antigen-impregnated artificial substrate can then be fixed and embedded identically to a piece of tissue. The BSA substrate can also be dried and then sectioned for immunolabeling with or without chemical fixation and without exposing the antigen to dehydrating agents and embedding resins. The effects of various fixation and embedding procedures can thus be evaluated separately. Other parameters affecting immunocytochemical labeling, such as antibody and conjugate concentration, can also be evaluated. We used this system, along with immunogold labeling, to determine quantitatively the optimal fixation and embedding conditions for labeling of hepatitis B surface antigen (HbsAg), human IgG, and horseradish peroxidase. Using unfixed and unembedded HBsAg, we were able to detect antigen concentrations below 20 micrograms/ml. We have shown that it is not possible to label HBsAg within resin-embedded cells using conventional aldehyde fixation protocols and polyclonal antibodies.

99mTc-labelled human serum albumin: Chemical and electrochemical labelling methods

The labelling of human serum albumin /HSA/ with99mTc has been investigated using a chemical method /stannous citrate/ and electrolytically generated tin/II/ ions. A comparative study of various chemical parameters and current intensities has been carried out in order to find the optimal conditions for labelling. The labelling yield was over 95%, for the chemical and electrolytical methods.

Radioimmunoassay for human osteocalcin using an antibody raised against the synthetic human (h37–49) sequence

Radioiodination of synthetic human 37-49 osteocalcin requires optimal labeling conditions in order to obtain a maximum of mono- and di-iodinated tracer with little contamination by tri- and tetra-iodinated products or "radio-damage." The antibody raised against osteocalcin(h37-49) had the highest affinity for the C-terminal peptide used for iodination and the larger peptide (h30-49). The intact bovine osteocalcin (b1-49) revealed less immunoreactivity. This C-terminal specific radioimmunoassay detected the intact human osteocalcin in HPLC purified plasma and peritoneal dialysate from patients with terminal renal insufficiency and in extracted human bone. Some quantities of osteocalcin peptides with a higher hydrophobicity were predominantly detected in uremic plasma. These peptides that had a higher molecular weight than the intact human molecule might represent aggregated forms of the intact bone-derived osteocalcin. Immunoreactivity in plasma samples from healthy individuals revealed a remarkable difference as to which substance was employed for anticoagulation. Compared to heparin, the addition of EDTA largely reduced the osteocalcin immunoreactivity, implying that conformational changes within the N-terminal portion (containing the Gla- and Cys-residues) are extended to the C-terminal portion.

Immunogold-silver staining of lymphocyte surface antigens on cells in suspension and in lymph node cryostat sections.

An immunogold-silver staining (IGSS) technique for the light microscopical detection of leucocyte cell surface antigens in cell suspensions and cryostat sections is described. The specimens were first incubated with monoclonal mouse antibodies and then with colloidal gold-labelled goat anti-mouse antibodies. They were then immersed in a physical developer, counterstained and mounted. In light microscopy, the tissue architecture and the cellular morphology were well preserved. Positive cells showed dark granules on their surface membranes. Optimal labelling conditions were determined. This method proved to be a reliable tool for the enumeration of T-cells and their subsets in peripheral blood. The dense labelling permitted the use of panoptic counterstains like May-Grünwald-Giemsa or Wright's stain. This IGSS technique was used to determine the distribution of the T- and B-cell subsets in cryostat sections of reactive lymph nodes. The sensitivity of the method was comparable with that of immunofluorescence microscopy for cell suspensions and that of the biotin-avidin-peroxidase technique for tissue sections. Immunogold-silver staining was combined with enzyme cytochemistry. In dark-field or epipolarization microscopy the labelling appeared as bright granules on a dark background. With its dense granular membrane labelling and its good morphology IGSS is an ideal method for the study of particular cell types in mixed cell suspensions. In addition, it could be a general method for the detection of cell surface antigens in all kinds of cells and tissues.

Detection of Cell Surface Antigens in Cryostat Sections with Immunogold-Silver Staining

Immunogold-silver staining was used for the detection of lymphocyte cell surface antigens in cryostat sections of lymphoid tissues. The sections were incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. They were then immersed in a physical developer, counterstained, and mounted. In light microscopy, the tissue architecture was well preserved, and a dark labeling was seen on the positive cells. Optimal labeling conditions were determined. The distribution of the lymphocyte subsets, as defined by a panel of monoclonal antibodies in tonsil and reactive lymph nodes, was similar to that found with a biotin-avidin-horseradish peroxidase method. The monoclonality of the neoplastic cells in lymph nodes of B-cell non-Hodgkin's lymphomas clearly could be demonstrated. The sensitivity of the technic was comparable with that of the biotin-avidin-horseradish peroxidase labeling method. In addition, immunogold-silver labeling was combined with acid phosphatase cytochemistry.

In vivo mobility of fatty acid end groups of Bacillus thuringiensis plasma membrane lipids during growth and sporulation.

The mobility of 13C specifically labeled branched chain end groups of iso-even fatty acids in intact, live Bacillus thuringiensis cells was studied by 13C nuclear magnetic resonance spectroscopy. This study apparently represents the first direct observation of branched chain carbon atoms in living cells. End groups were labeled using DL-[beta, delta, delta'-13C]valine as a precursor chain initiator for iso-even fatty acid synthesis after using L-[delta, delta'-14C]L-valine to determine optimal conditions for labeling of the membrane fatty acid end groups. Cell survival in the NMR was determined for various lengths of time at 28 and 39 degrees C. Subsequently, 13C-labeled vegetative cells, sporulating cells (three stages of development), and purified mature spores were analyzed by 13C NMR using corresponding unlabeled cells as controls. Spin lattice relaxation times (T1) were obtained for the enriched iso-branched region at 23.3 ppm and for the natural abundance peak for the glycerol backbone (carbons 1 and 3) of the membrane lipids at 61.7 ppm. The T1 of the glycerol carbons (0.08 s) did not change significantly with stage of development or temperature. The T1 of the iso-even enriched end group changed dramatically from vegetative cells (0.70s) to sporulating cells (0.28 s) at 28 degrees C. A decrease in the T1 was also observed at 39 degrees C from 0.91 s for vegetative cells to 0.54 s for sporulating cells. Accompanying the reduced mobility indicated by the T1 values, there was a general decline in the signal-to-noise ratios of identically acquired spectra as sporulation continued which culminated in the lack of discernible plasma membrane lipid resonances in purified mature spores. The progressive loss of signal appeared to have resulted from a continuous decline in the fraction of plasma membrane fatty acids with sufficient mobility to give signals above background.

Dopamine receptors in the rat brain: quantitative autoradiographic localization using [ 3H]sulpiride

In vitro labeling of tissue sections with [ 3H]sulpiride has been utilized in the present study to autoradiographically localize D 2-dopamine receptors in the rat brain. Preliminary biochemical studies, using slide-mounted tissue sections, were performed to define the optimal labeling conditions for this binding. Autoradiograms were generated by apposition of the labeled tissue sections to tritium-sensitive film. Specific binding sites for [ 3H]sulpiride were localized to the caudate-putamen, nucleus accumbens, olfactory tubercle, glomerular layer of the olfactory bulb, pituitary, laminae I and III of the entorhinal cortex, substantia nigra, lateral mammillary nucleus and the stratum-lacunosum moleculare of the hippocampus. The high selectivity of [ 3H]sulpiride for the D 2-dopamine receptor indicates that it is a valuable tool for the autoradiographic localization and quantitation of neuroleptic receptors.

Direct comparison of the rates of internalization and degradation of covalent receptor-insulin complexes in 3T3-L1 adipocytes. Internalization of occupied receptors is not the rate-limiting step in receptor-hormone complex degradation.

Insulin receptors on the surface of 3T3-L1 adipocytes were photolabeled using the iodinated analog, B29-lysine-substituted N-[N'-(2-nitro-4-azidophenyl)glycyl]insulin. Under optimal labeling conditions (below 15 degrees C), greater than 95% of the labeled receptor remained on the cell surface prior to incubation at 37 degrees C. When the labeled monolayers were returned to their normal culture environment (37 degrees C), the covalent receptor-insulin complexes were rapidly internalized at initial rates equivalent to 130-170% of labeled surface receptor/h. Internalization of the complexes proceeded to an equilibrium or end point distribution of 40% internal receptor and 60% cell-surface receptor. Under the several labeling conditions tested, covalent receptor-insulin complexes were degraded in an apparent first order process at 37 degrees C with half-lives between 5 and 7 h. This rate was equivalent to only 10% of the labeled receptor being degraded per h and was 13-17-fold slower than the initial rate of labeled receptor internalization. This study directly demonstrates that the initial rate of internalization of covalent receptor-insulin complexes is not the rate-limiting step in their degradation in 3T3-L1 adipocytes. Furthermore, 3T3-L1 adipocytes are unable to internalize all of the labeled surface receptor, suggesting that two classes of internalization competent and incompetent receptor may exist or that an equilibrium distribution of internal and cell-surface receptor is established by the relative rates of internalization and recycling of labeled receptor.

Optimal Conditions for the Preparation of Ferritin-labeled Antibodies Defined by Binding to Their Antigen in an ELISA

An ELISA with covalently fixed antigens was used to measure and define ferritin-labeled antibodies. In the presented model, RIG covalently fixed to glass tubes served as antigen. Twenty ferritin-labeled SWaRIG conjugates were prepared with different molar ratios of antibody: ferritin: glutaraldehyde. At various dilutions, their ability to react with the antigen was compared. The amount of FRT in the reactive conjugates was measured using alkaline phosphatase-labeled antibodies against ferritin. The covalent binding of antigens to glass surfaces resulted in a low unspecific binding as shown previously. Besides, these glass tubes with antigens immobilized on their inner surface could be used more than once. This aspect of it renders this test particularly suitable for systems where rare or expensive antigens are used. The range of O.D. values reflecting the amount of reactive SWaRIG/FRT detected in 20 different conjugates (dil 1:1000) was spread over a good range which allowed a specification of optimal conditions for FRT labeling of antibodies. It is concluded that in addition to an electron microscopic evaluation of the conjugates, this assay may be very helpful in defining optimal conditions for the coupling procedure.

Efficient translation and polyribosome binding of 125I-labelled rabbit globin messenger ribonucleoprotein

Rabbit polyribosomal globin messenger ribonucleoprotein (mRNP) was labelled under mild conditions, using 125I and Iodogen, in the protein moiety so that the fate of mRNA-associated proteins could be followed during translation. 125I-mRNP was shown to retain functional activity in the nuclease-treated reticulocyte lysate translation system under optimal labelling conditions. Polyribosome binding of 125I-mRNP and its sensitivity to cycloheximide indicated a functional- and translation-dependent binding of mRNP proteins. The results constitute a successful and direct approach to the study of mRNA-associated proteins in translational control.

Indium-111 tropolone, a new tracer for platelet labeling.

Platelets have been labeled with a new neutral, lipid-soluble metal complex of indium 111 (111In) and tropolone. Unlike oxine, which is soluble in ethyl alcohol, tropolone is soluble in isotonic saline. Platelet labeling with 111In tropolone can be performed in both acid-citrate-dextrose (ACD) plasma and ACD saline within two hours. Labeling efficiency has been 80% to 90%. 111In tropolone in ACD saline and ACD plasma at tropolone concentrations of 5 and 10 micrograms/ml, respectively, and incubation of the platelets with the tracer at room temperature for 20 minutes were optimal conditions for labeling. The authors have developed an ACD-saline kit for convenient preparation of 111In-labeled platelets. No adverse effect of 111In tropolone on platelets has been observed in studies of biodistribution, recovery, and survival of platelets in rabbits and dogs.

Exogenous dTMP utilization by a novel tup mutant of Saccharomyces cerevisiae.

The rate and extent of entry of dTMP were measured in strains of Saccharomyces cerevisiae carrying two new tup mutations (tup5 and tup7) and most of the other tup mutations which have been reported previously by others. The tup7 mutation allowed dramatically greater accumulation of dTMP than any of the other mutations tested. Specific labeling of DNA by [CH3-3H]dTMP, fate of the dTMP pool inside of the cells, and degradation of the dTMP in the culture medium were investigated in strains carrying the tup7 mutation. The extracellular dTMP was not appreciably degraded, and that accumulated intracellularly was readily phosphorylated to dTDP and dTTP. Under optimum labeling conditions, 60 to 80% of the total thymidylate residues in newly synthesized DNA were derived from the exogenously provided dTMP, even in the absence of a block in de novo dTMP biosynthesis. An apparent Km for entry of 2 mM dTMP was found. The tup7 mutation increased permeability to dTMP (and some other 5'-mononucleotides), but did not affect uptake of nucleosides and purine and pyrimidine bases. Uptake of dTMP could be almost completely inhibited by moderate concentrations of Pi. These findings and other observations suggest that entry of dTMP in strains carrying the tup7 mutation is mediated by a permease whose function in normal cells is the transport of Pi.

Immunoelectron microscopic studies of Actinomyces viscosus.

Optimal conditions for labelling Actinomyces viscosus (T14V) in thin sections by an indirect immunoperoxidase technique are described. The influence of several fixatives on the degree of labelling of bacterial cells was tested in pure cultures and in dental plaques. Some pure cultures were also processed without prior fixation. The effect of different incubation conditions on the degree of labelling was evaluated in pure cultures. The optimal labelling conditions included fixation with paraformaldehyde (4%) plus glutaraldehyde (0.25%), incubation with highly diluted rabbit anti‐T14V γglobulin for 24 hours at 4°C, followed by incubation with diluted sheep anti‐rabbit IgG conjugated to horseradish peroxidase, and subsequent histochemical visualization of the peroxidase. Optimal labelling conditions were used to check for cross‐reactions between the anti‐T14V γ‐globulin and some common oral bacteria. The well‐known cross‐reaction with Actinomyces naeslundii was noted, as well as a slight cross‐reaction with Bacterionema matruchotii.Optimal conditions were used for labelling sections of dental plaques. Wide variations were observed in the number of labelled cells. Cells labelled with anti‐T14V γ‐globulin were unlabelled when consecutive sections of these cells were treated with control normal rabbit serum γ‐globulin or anti‐Streptococcus mutansγglobulin. Cells labelled with anti T14V γglobulin tended to be cocco‐bacillary in shape in the superficial layers of plaque. The deeper layers exhibited more filamentous forms. These observations are compatible with the known pleomorphism of A. viscosus in vitro.

10] Sulfhydryl modification and labeling of myosin

Publisher Summary This chapter discusses the sulfhydryl modification and labeling of myosin. Two sulfhydryl groups, the SH 1 and SH 2 groups located on the heavy chain of subfragment-1 of myosin, have been extensively studied because their modification was found to affect dramatically the ATPase activities of myosin. The sulfhydryl reagents differ in their reactivity toward the SH 1 groups on myosin. Consequently, for each reagent it is desirable to establish optimal conditions for specific labeling of the SH 1 groups. This can be done by following the changes in myosin ATPases as a function of increasing incorporation of the reagent. The goal in such experiments is to find reaction conditions under which incorporation of 1 mol of reagent per mole of myosin head results in maximum inhibition of K(EDTA)-ATPase and maximum activation of the Ca- ATPase. These changes in myosin activity are indicative of SH1 modification. The assignment of the modification site on the basis of ATPase properties alone is purely inferential. It is based on the previous identification of the modified peptides in reactions of myosin with NEM and iodoacetamide. Addtionally, specific and relatively rapid modification of the SH 1 groups on myosin can be achieved by allowing N-ethylmaleimide and maleimide-based reagents to react with myosin, S-l, or heavy meromyosin.

3H]Spiperone binding sites in brain: autoradiographic localization of multiple receptors

[ 3H]Spiperone ([ 3H]SP) binding sites were localized by light microscopic autoradiography, after in vitro labeling. The kinetic and pharmacological characteristics of these binding sites were studied in slide-mounted sections of rat forebrain, and optimal labeling conditions were defined. Autoradiograms were obtained by apposing emulsion-coated coverslips to labeled sections. Diffetrential drug sensitivity allowed the selective displacement of [ 3H]SP from dopamine receptors by ADTN, from serotonin receptors by cinanserin, from both by haloperidol and from unique spiperone sites by unlabeled spiperone. The various sites presented a differential anatomical localization. For example, only dopaminergic sites were found in the glomerular laver of the olfactory bulb; only serotonergic sites were found in lamina IV of the neocortex, and a high concentration of unique spiperone sites was found in parts of the hippocampus.

A chemical method for the labeling of fibrinogen with 99mTc

A new method allowing the labelling of fibrinogen with 99mTcO4- is described. The 99mTcO4- is reduced by stannous chloride in an alkaline medium. After 1 h incubation with fibrinogen, the pH was brought down to 7.1. The study of (1) the pH of the solution, (2) the quantity of the reducer, and (3) the time of incubation allowed us to specify optimal conditions for labeling. The labeling yield varied from 91.68% plus or minus 6.05% to 95.18% plus or minus 2.13% according to the method of control used: the precipitation of fibrinogen by (NH4)2SO4 25% or thin-layer chromatography with methylethylketone as solvent. Clottable radioactivity averaged 72% plus or minus 4.43%. Column chromatography separated the tracer into two radioactive peaks. The first peak corresponded to the fibrinogen and carried 73.36% plus or minus 5.8% of the total radioactivity. The total recovered radioactivity amounted to 89.86% plus or minus 8.07%. Spectroscopic clottability was 88.23% plus or minus 2.42%. The in vivo stability of the labeling and the molecule was followed for 24 h after intravenous injection. If the radioactivity measured in the 5 min sample was considered to equal 100%, the 24 h sample averaged 32.71% plus or minus 3.25%, of which 85.68% plus or minus 2.92% was recovered in the clot. In conclusion, this method enabled us to obtain a stable tracer that we used for routine investigations in man.

Methods for estimating the concentration of different forms of androgen receptor in rat ventral prostate

Glycine-Sepharose 4B, a new material for chromatography, and DNA cellulose have been evaluated for use in estimating the concentration of specific forms of androgen receptor in rat ventral prostate. Chromatography with either material facilitates the partial isolation of more than a single type of cytoplasmic receptor but only one form is recovered in substantial amounts. The predominance of this form results from the greater efficiency of receptor labeling afforded by the use of tissue minces. Both cytoplasmic and nuclear forms of receptor bind to glycine-Sepharose 4B, but only the former binds to DNA cellulose in appreciable amounts. Under optimal labeling conditions glycine-Sepharose 4B chromatography yields a more accurate estimate of the concentration of receptors per cell, namely 12,000 molecules in the nucleus before castration, and 14,000 molecules in the cytoplasm 1 day after castration. The findings are consistent with the idea that nuclear receptor is discharged into the cytoplasm upon acute withdrawal of androgen.

The influence of experimental conditions on the efficiency of labeling human serum albumin with 99 mTc, using Sn(II) as the reductant

The influence of the pH and of the albumin and Sn(II) concentrations on the efficiency of labeling human serum albumin with 99 m Tc has been investigated by means of a 4×3×3 factorial design, with duplication. The equation, describing the response surfaces as a function of the three investigated variables, has been derived. This response equation is used to find the optimum labeling conditions and in a discussion of the chemistry of the labeling.

Tritium labeling of immunoglobulin with iodoacetic acid

The labeling of immunoglobulin with iodoacetic acid-2- 3H has been found to be markedly dependent on temperature, pH and time. The optimum labeling conditions combining maximum specific radioactivity with minimum alteration to the antigen binding ability of the labeled immunoglobulin were 70°C and pH 9·8 for 30 min. This resulted in labeled immunoglobulin whose specific radioactivity ranged between 2700 and 3400 dpm/μg and 45 per cent of which could bind to the homologous antigen. Bands obtained by the SDS polyacrylamide gel electrophoresis of the labeled preparation were strictly comparable to those of unlabeled immunoglobulin and the protein specific radioactivity was found to be largely confined to polypeptides of molecular weight greater than 1·6 × 10 5 daltons.