Biomedical subjects

V M Mukkala

Publications and source records attributed to V M Mukkala.

15 recordsLinked to original sources

PubMed2000

Production and characterisation of monoclonal antibodies against a very small hapten, 3-methylindole.

Monoclonal antibodies were produced against a very small (131.2 Da) hapten, 3-methylindole. Nine derivatives of 3-methylindole were synthesised with spacers ending in a carboxyl group, and coupled to immunogenic carriers and europium chelate labels. Almost all the antigens elicited an antihapten response, but the majority of the mAbs produced strongly recognised the spacer group and did not bind free 3-methylindole. However, specific antibodies were obtained with five immunogens. Specificity could be directed against the pyrrole ring by locating the bridging group to the aromatic moiety of the indole ring system. Any modification in the position 3 of the indole ring strongly hindered mAb binding to the compound, and the cross-reactivity of physiologically important compounds, such as tryptophan and tryptamine, was negligible for all of the mAbs. The developed hapten structures successfully focused antibody recognition to the important sub-determinants in the indole ring system. Similar constructs could also be useful in the development of antibodies against other indolic compounds.

Antibodies, Monoclonal↗

PubMed1997

Sensitive bioaffinity assays with individual microparticles and time-resolved fluorometry.

Future immunoassays and nucleic acid hybridization assays will be performed in miniaturized formats that utilize microchips or microparticles. This will require a sensitive detection technology that allows spatial resolution. By using fluorescent europium chelates and time-resolved microfluorometry, one can detect 11,000 europium molecules on individual microparticles. In a miniaturized noncompetitive immunoassay of prostate-specific antigen (PSA), we quantitatively detected 5 ng/L (0.05 amol per particle) of the analyte on an individual microparticle with excellent precision over the whole measurement range (CV <10%). Using a hybridization assay, we also could detect the deltaF508 mutation for cystic fibrosis on individual microparticles. Consequently, fluorescent lanthanide chelate labels and time-resolved microfluorometry qualify as the next generation of technology in this field.

Chelating Agents↗

PubMed1996

Time-resolved fluorometric assay for natural killer activity using target cells labelled with a fluorescence enhancing ligand.

A time-resolved fluorometric assay for the measurement of natural killer cell activity against target cells labelled with the acetoxymethyl ester of the fluorescence enhancing ligand 2,2':6',2"-terpyridine-6,6"-dicarboxylic acid (TDA) is described. The hydrophobic esterified form of TDA (bis(acetoxymethyl) 2,2':6',2"-terpyridine-6,6"-dicarboxylate, BATDA) diffuses readily through the cell membrane of viable cells. BATDA is hydrolysed by intracellular esterases resulting in accumulation of membrane impermeable TDA inside the target cells. After incubation of labelled K-562 cells with effector cells the TDA released from lysed cells into the supernatant is chelated with Eu3+. The natural killer cell activity is then quantified by measuring the intense fluorescence of the EuTDA chelates formed. Target cells are rapidly labelled when incubated with BATDA, TDA is released from target cells faster than 51Cr, the spontaneous release permits a short-term release assay to be set up and the detection of EuTDA is fast (5 min/96 well plate). Furthermore, this non-radioactive method permits the use of complex culture media since, in contrast to methods based on prompt fluorometry, the problem with autofluorescence can be avoided by the use of time-resolved fluorometry.

2,2'-Dipyridyl↗

PubMed1994

Solid-phase synthesis of chelate-labelled oligonucleotides: application in triple-color ligase-mediated gene analysis.

Oligonucleotides labelled with detectable groups are essential tools in gene detection. We describe here the synthesis of pyrimidine deoxynucleotide-building blocks, modified at their C-5 position with a protected form of a strongly chelating agent. These reagents can be used to introduce multiple metal ions into oligodeoxynucleotides during standard oligonucleotide synthesis. The chelating functions form strongly fluorescent complexes with europium ions, characterized by a wide separation between the excitation and emission spectra. Moreover, the long decay time of the fluorescence permits sensitive time-resolved fluorescence measurements. The chelates also have the stability required to function in triple-color assays involving europium, samarium, and terbium ions. We demonstrate the application of these reagents for ligase-based gene analysis reactions.

Base Sequence↗

PubMed1993

Di- and tetracarboxylate derivatives of pyridines, bipyridines and terpyridines as luminogenic reagents for time-resolved fluorometric determination of terbium and dysprosium.

A group of compounds composed of pyridine, 2,2'-bipyridine and 2,2':6',2''-terpyridine as the energy absorbing, triplet sensitizing moieties and two or four carboxylic acids as the chelating groups were synthesized and their ability to enhance terbium ion luminescence was elucidated. The dicarboxylic acid derivatives form highly luminescent Tb3+ chelates with luminescence quantum yields approaching 50%. Additional groups, such as methoxy groups or additional pyridine rings in the aromatic structure can be utilized to shift the excitation maxima towards longer wavelengths in order to produce more practicable excitation for practical use. The ligands giving the highest Tb3+ emissions were further evaluated in a two-step fluorescence enhancement system constructed for specific binding assays based on the simultaneous use of three or four ions as the labels (Eu3+, Tb3+, Sm3+ and Dy3+). In the two-step procedure, the first step comprises dissociative fluorescence enhancement based on an acidic solution of an aromatic beta-diketone used as the luminogenic ligand, whereafter Eu3+ and Sm3+ are quantitated. In the second step, the lanthanide ions form new chelates with the added ligands. These ligands are able to transfer the excitation energy they have absorbed with high efficiency to the chelated ions and produce high ion luminescence for Tb3+ and Dy3+.

2,2'-Dipyridyl↗

PubMed1992

Detection of amplified HTLV-I/-II viral sequences using time-resolved fluorometry.

Since its discovery, the polymerase chain reaction (PCR) has been used for different purposes in the field of DNA research. We tested the PCR for the diagnosis of HTLV-I/-II infections. PCR was used to amplify 141- and 149-base pair regions from the HTLV-I and HTLV-II virus genomes, respectively. The annealing temperature in the PCR amplification was optimized using 20% polyacrylamide gels and silver staining. Even a slight change (3 degrees C) in the annealing temperature had an effect on the specificity of the reaction. The PCR products were detected with biotin and Eu-labeled oligonucleotide probes in a solution hybridization format. The linearity of the assay was tested with serial dilutions of purified chromosomal DNA containing integrated HTLV-II sequences. The linearity was found to be dependent on the number of cycles used in the PCR amplification. The best linearity, at a target level of a few copies, was achieved using a low number of cycles. The specificity of the assay was tested using HTLV-I and HTLV-II-infected lymphocytes from the cell lines Hut102 and MO480, respectively. No cross reactivity between these analytes was observed.

Base Sequence↗

PubMed1991

The use of europium (Eu3+) labelled primers in PCR amplification of specific target DNA.

The polymerase chain reaction (PCR) has many potential applications in the field of DNA probe diagnostics. Here we describe a method that utilizes PCR and time-resolved fluorometry (TRF) for the detection of specific target DNA. First the DNA segment to be detected is amplified according to standard procedures. Then a pair of europium (Eu3+) and biotin-labelled primers nested within the amplified fragment is incorporated in a few additional PCR cycles. Thus amplified DNA fragments are generated that contain an affinity label (biotin) and a detectable label (europium). The doubly-labelled amplified DNA fragments are collected onto streptavidin coated microtitration strips and the bound Eu3+ is measured in a time-resolved fluorometer. We show here the application of this method to the detection of HIV-1 DNA. As few as five copies of HIV-1 DNA could readily be detected using this assay. The method described here is sensitive, rapid and easy to employ. In addition it lends itself to automation.

Bacterial Proteins↗

PubMed1991

Co-fluorescence of europium and samarium in time-resolved fluorimetric immunoassays.

In the presence of an excess of Y3+, the fluorescence intensities of Eu3+ and Sm3+, chelated with benzoyltrifluoroacetone (BTA) or thenoyltrifluoroacetone (TTA) in an aqueous solution containing 1,10-phenanthroline, were increased by factors ranging from 209- to 811-fold. This co-fluorescence phenomenon was used in a highly sensitive time-resolved fluorimetric detection of the lanthanides, Eu3+ and Sm3+. The detection limits of Eu3+ in the BTA- and TTA-based solutions were 4 and 15 fmol dm-3, respectively. The detection limits of Sm3+ were 0.11 and 0.12 pmol dm-3, respectively. The co-fluorescence enhancement systems were also applied in the double-label time-resolved fluorimetric immunoassay of luteinizing hormone and follicle stimulating hormone using specific antibodies labelled either with Eu3+ or Sm3+. The co-fluorescence enhancement solution was superior as compared with the commercial 'direct' fluorescence enhancement solution based on the acidic solution of beta-naphthoyltrifluoroacetone, trioctylphosphine oxide and Triton X-100, in respect to the signal level obtained and the sensitivity. It is suited to time-resolved fluorimetric immunoassays in which particularly high detection sensitivities are required, and it can also be used in double-label assays employing Eu3+ and Sm3+ chelate labels.

Europium↗

PubMed1989

The synthesis and use of activated N-benzyl derivatives of diethylenetriaminetetraacetic acids: alternative reagents for labeling of antibodies with metal ions.

A series of bifunctional chelating agents--substituted benzyl derivatives of diethylenetriaminetetra acids--was synthesized. These agents were used to label antibodies with a lanthanide, Eu3+. The stabilities of their antibody conjugates were evaluated under different conditions and the dissociation rates of Eu3+ were measured at pH 3.2, which is used for fluorescence enhancement in time-resolved fluoroimmunoassays. The synthesized complexing agents were also compared to other reagents used for metal labelings, to diethylenetriaminepentaacetic acid-dianhydride, and to p-isothiocyanatophenyl-EDTA. The asymmetric p-isothiocyanatobenzyl derivative of diethylenetriaminetetraacetic acid-Eu3+ showed reasonably good stability at neutral pH but released Eu3+ rapidly in the acidic fluorescence enhancement solution. This makes it an optimal choice for chelate labeling in dissociation-based time-resolved fluoroimmunoassays.

Animals↗

PubMed1988

Lanthanide chelates as new fluorochrome labels for cytochemistry.

Anti-rabbit IgG labeled with a new fluorescent europium chelate was used to localize rabbit IgG to human smooth muscle myosin in a histological section. The antibody labeled with the europium chelate could be viewed with a conventional fluorescence microscope with a steady-state light source. This result encourages the development of a time-resolved fluorescence microscope, because a significant improvement in the signal-to-noise ratio can be anticipated.

Fluorescent Antibody Technique↗

PubMed1984

Europium as a label in time-resolved immunofluorometric assays.

A nonisotopic immunoassay has been developed based on a sensitive detection of europium (III) in water solution using time-resolved fluorometry. The europium label is bound to the antibody with EDTA derivatives, either diazophenyl-EDTA-Eu or isothiocyanatophenyl-EDTA-Eu. After the immunometric assay has been completed the europium is preferably dissociated from the antibody at low pH and measured by time-resolved fluorescence in a micellar solution containing Triton X-100, beta-diketone, and a Lewis base. The detergent solubilizes the chelating compounds in the solution and excludes water from the fluorescent ligand-europium complex. Europium concentrations as low as 5 X 10(-14)M were measured using a 1-s counting time. The sensitivity of the immunoassay of rabbit IgG used as a model system was 25 pg/ml (6 pg/assay).

Animals↗

PubMedDate not supplied

A time-resolved study of the mechanism of the energy transfer from a ligand to the lanthanide(III) ion in solutions and solid films.

The photochemical properties of some lanthanide chelates developed for immunohistochemistry have been studied in water and in solid Langmuir-Blodgett films. The fluorescence and triplet-state lifetimes of 4-(phenylethynyl)pyridine (PET), di[(phenylethynyl)pyridine] (D-PET), phenylterpyridine (PTP) and their tetra- or penta-acid derivatives (-TA or -PA) were measured in the presence and absence of Gd(III)-, Tb(III)- and Eu(III)-ions. The mechanism for the total process and the rate constants and quantum yields for the individual reaction steps and for the total process were determined in water solution. Time-resolved absorption and luminescence methods were also used to study the energy transfer between an amphiphilic 4-[4-[(C(10)H(12))(2)NCO]phenylethynyl]-pyridine tetra acid (A-PET-TA) and the Tb(III)- and Eu(III)-ions in solid Langmuir-Blodgett films. Luminescence and transient absorption rate constants were determined.

Chelating Agents↗

PubMedDate not supplied

Europium-labeled oligonucleotide hybridization probes: preparation and properties.

A chemical method for labeling of oligonucleotide probes with europium chelates is presented. A modified deoxycytidine phosphoramidite is used to introduce multiple reactive amino groups to the oligonucleotide during the synthesis phase. Upon deprotection and purification of the modified oligonucleotide, an isothiocyanate derivative of a stable Eu chelate is reacted with the primary amino groups. The labeling technology enables the coupling of a high number of Eu chelates to a single probe. The melting temperatures and hybridization efficiencies of the oligonucleotides are not significantly altered by the labeling process. However, hybridization kinetics of the oligonucleotides are affected by the introduction of multiple modified deoxycytidine residues. In a solid-phase hybridization assay up to 10(7) target molecules can be detected.

Base Sequence↗

PubMedDate not supplied

Synthesis of europium(III) chelates suitable for labeling of bioactive molecules.

Two different kinds of europium(III) chelates, luminescent and nonluminescent, were prepared. The chelates were coupled to bioanalytical reagents, such as antibodies, after activations of the amino group on the chelates with thiophosgene,2,4,6-trichloro-1,3,5-triazine, or iodoacetic anhydride. The reactivities of the activated luminescent chelates in the labeling of antibodies as well as the effects of both the coupling ratio and the linkage group to the luminescence quantum yield of the antibody-bound chelate were studied in aqueous buffer solution.

Animals↗

PubMedDate not supplied

Influence of coupling method on the luminescence properties, coupling efficiency, and binding affinity of antibodies labeled with europium (III) chelates.

A series of chelating 4-(phenylethynyl)pyridines having various 1,3,5-triazin-2-ylamino groups at the para position of the phenyl ring was synthesized. Their europium chelates were coupled to antibodies and the properties of antibody conjugates analyzed by fluorometry and in time-resolved fluorometric immunoassay. The substituents in the triazine ring were observed to have various effects on the chelate luminescence, the labeling properties of the chelates, and the immunoreactivity of labeled antibodies. The series of substituted triazinyl derivatives serves as a model of bioreactive groups that can be applied when certain properties are searched for, such as improved chelate solubilities, minimized internal quenching, different effects on the ligand triplet state, and stipulated coupling reactivities.

Antibodies↗