Cell-Dependent Chemiluminescence. Modulation of the N-Formyl Chemotactic Peptide (Fnlpntl) Mediated Oxidative Burst in Human Polymorphonuclear Leukocytes (Pmnl) By Murine Monoclonal Antibody Nms-1

Binding of purified monoclonal antibody (moAB) IgM NMS-1 to suspended initially spherical living human PMNLs is not associated with the generation of chemiluminescence but was found to enhance the chemiluminescence response to the N-formyl chemotactic peptide FNLPNTL.

We investigated quantitatively the kinetics of oxygen metabolite generation by PMNLs stimulated with FNLPNTL ± moAB NMS-1 using luminol-dependent chemiluminescence as a very sensitive detection system. Chemiluminescence detection allowed the analysis of the time sequence of onset and development of reactive oxygen metabolites following stimulation of PMNLs by FNLPNTL in the presence of moAB NMS-1. The increase of response of PMNLs stimulated with FNLPNTL in the presence of moAB NMS-1 depended on the concentration of the antibody and the sequence of stimulus addition.

Stimulation of human PMNLs by 10nM FNLPNTL induced a rapid burst of chemiluminescence which peaked ∼5min after stimulus addition. The subsequent addition of moAB NMS-1 (≥2μg/ml DPBS(+)—0.1% HSA, 37°C) to FNLPNTL-stimulated PMNLs—after the FNLPNTL-mediated response had already decayed (16-18 min) - without delay induced a second burst of oxygen metabolite generation. The magnitude of this second peak of activation was dose-dependent.

Treatment of PMNLs with moAB NMS-1 (≥ 1μg/ml DPBS(+)—0.1% HSA, 3 min, 37°C)—prior to FNLPNTL (10nM) stimulation - increased rate and magnitude of the FNLPNTL-mediated response. This response is biphasic with the first peak at the FNLPNTL position and a second, higher peak ∼16 min after FNLPNTL addition. The magnitude of response was dose-dependent. The latency (lag time) of the respone was not changed compared to controls which received no moAB NMS-1 treatment.

The observed moAB NMS-1 dependent increase in FNLPNTL-mediated chemiluminescence is transient (5-60 min), persistent activation was not detected.     

Optimization of an HPLC peroxyoxalate chemiluminescence detection system for some dansyl amino acids

Bis(2,4,6-trichlorophenyl) oxalate (TCPO)-hydrogen-peroxide-generated chemiluminescence (CL) of four dansyl amino acids has been used as a model system for the optimization of a detection system in reversed-phase high-performance liquid chromatography. Dansylated alanine, glutamic acid, methionine, and norleucine were subjected to peroxyoxalate induced CL in a static system and in a flow system under various conditions with respect to TCPO (ethyl acetate) and hydrogen peroxide (acetone) concentrations, solvent composition and flow, using a two-pump or a one-pump post-column reagent system. From the CL-decay curve, the influence on the emission signal from the total flow rate in the detector was investigated. Special attention was focused on the mixing of the LC eluate and the reagent in order to combine an efficient collection of the emitted light using a 74μI flow cell (originally 10μI in the fluorescence detector) with minimal extra column band broadening. Therefore, a capillary fused-silica tubing of about 100μm i.d. was inserted against the end-frit of the column and brought through a mixing tee, in which the solutions of TCPO and hydrogen peroxide were added. The column end tubing ended in the flow cell and the LC eluate and the reagents were mixed when entering the flow-cell. Average detection limits (S/N=2) of 200fmol injected dansylated amino acid could be reached. A comparison is made between the use of TCPO and DNPO (bis (2, 4-dinitrophenyl) oxalate).     

Cell-Dependent Chemiluminescence. Modulation of the N-Formyl Chemotactic Peptide (Fnlpntl) Mediated Oxidative Burst in Human Polymorphonuclear Leukocytes (Pmnl) By Murine Monoclonal Antibody Nms-1

Binding of purified monoclonal antibody (moAB) IgM NMS-1 to suspended initially spherical living human PMNLs is not associated with the generation of chemiluminescence but was found to enhance the chemiluminescence response to the N-formyl chemotactic peptide FNLPNTL.

We investigated quantitatively the kinetics of oxygen metabolite generation by PMNLs stimulated with FNLPNTL ± moAB NMS-1 using luminol-dependent chemiluminescence as a very sensitive detection system. Chemiluminescence detection allowed the analysis of the time sequence of onset and development of reactive oxygen metabolites following stimulation of PMNLs by FNLPNTL in the presence of moAB NMS-1. The increase of response of PMNLs stimulated with FNLPNTL in the presence of moAB NMS-1 depended on the concentration of the antibody and the sequence of stimulus addition.

Stimulation of human PMNLs by 10nM FNLPNTL induced a rapid burst of chemiluminescence which peaked ～5min after stimulus addition. The subsequent addition of moAB NMS-1 (?2μg/ml DPBS(+)—0.1% HSA, 37°C) to FNLPNTL-stimulated PMNLs—after the FNLPNTL-mediated response had already decayed (16-18 min) – without delay induced a second burst of oxygen metabolite generation. The magnitude of this second peak of activation was dose-dependent.

Treatment of PMNLs with moAB NMS-1 (? 1μg/ml DPBS(+)—0.1% HSA, 3 min, 37°C)—prior to FNLPNTL (10nM) stimulation – increased rate and magnitude of the FNLPNTL-mediated response. This response is biphasic with the first peak at the FNLPNTL position and a second, higher peak ～16 min after FNLPNTL addition. The magnitude of response was dose-dependent. The latency (lag time) of the respone was not changed compared to controls which received no moAB NMS-1 treatment.

The observed moAB NMS-1 dependent increase in FNLPNTL-mediated chemiluminescence is transient (5–60 min), persistent activation was not detected.     

The increasing effect of some synthetic peptides on luminol-dependent chemiluminescence of mouse blood

Some synthetic peptides increased the luminol-dependent chemiluminescence of mouse blood during phagocytosis. It is suggested that the levels of antimicrobial activity of the neutrophil peroxidase system can be raised very quickly (within some dozen of seconds) by these peptides. This raises the possibility of finding a new approach to the therapy for infectious diseases.     

Magic lite design and development

Chemiluminescence immunoassays have now achieved a recognized place in the diagnostic laboratory. The advantages of this non-isotopic technology derive from the use of acridinium esters which can be used to label antigens and antibodies to high specific activities, as well as from optimized immunochemistry. The availability of simple, reliable instrumentation for chemiluminescence measurement together with a range of assay kits offers a logical alternative to traditional radioimmunoassay.     

Determination of H2O2-dependent generation of singlet oxygen from human saliva with a novel chemiluminescence probe

Singlet oxygen (¹O₂) has been shown to play an important role in salivary defense system, but its generation process and level from human saliva remain uncertain due to the lack of a reliable detection method. We have previously reported 4,4′(5′)-bis[2-(9-anthryloxy)ethylthio]tetrathiafulvalene (BAET) as a novel chemiluminescence probe for ¹O₂. In this work, the probe is successfully used to characterize H₂O₂-dependent generation of ¹O₂ from saliva in real time. However, the yield of ¹O₂ is found to be very low, for example, being about 0.13 nmol from 200 μL saliva in the presence of 1 mM of hydrogen peroxide over a 5-s reaction period. The result is also compared with that obtained with another ¹O₂ probe 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA), demonstrating that, besides ¹O₂, the other reactive oxygen species such as hydroxyl radical may also be involved in the reaction of saliva with H₂O₂. Furthermore, the present study shows that the selectivity of BAET for ¹O₂ is much higher than that of CLA and thus BAET is highly suited for the detection of ¹O₂ in the presence of other reactive oxygen species in biological systems.     

An assay for the determination of biologically active bone morphogenetic proteins using cells transfected with an inhibitor of differentiation promoter-luciferase construct

Bone morphogenetic proteins (BMPs) control cell fate by regulating gene expression, especially inhibitor of differentiation (Id) genes. This property has been exploited to create a highly sensitive assay for quantification of active BMP. Embryonic mouse cells (C3H10T1/2) were stably transfected with an expression construct (BRE-Luc) containing a BMP-responsive element fused to the firefly luciferase reporter gene. BRE results from a multimerization of distinct sequences elements from a mouse Id1 promoter [15]. The addition of BMP-2 (0.5-100ng/ml) to the transfectants resulted in a dose-dependent increase in luciferase activity in the cell lysates. This new assay was 100-fold more sensitive than the classical alkaline phosphatase (ALP) activity assay (0.5-1 vs. 50-100ng/ml, respectively) as well as much more rapid (24h vs. 3-6 days, respectively, of BMP treatment). This new assay is specific to BMPs (BMP-2, BMP-4, and BMP7) as evidenced by its relative insensitivity to TGFbeta1, bFGF, and VEGF. Because of its BMP specificity, this rapid, sensitive, nonradioactive, and easily performed assay could be used in monitoring the biological activity of BMP and, eventually, as a cell-based screening assay to identify and evaluate molecules that modulate BMP signaling in cells.     

Enhancement of chemiluminescence for vitamin B12 analysis

In the current article, chemiluminescence (CL) from the vitamin B₁₂ and luminol reaction was studied under alkaline conditions to develop a sensitive analytical method for vitamin B₁₂ using the carbonate enhancement effect. The method was successfully applied to the determination of vitamin B₁₂ in vitamin B₁₂ tablets, multivitamin capsules, and vitamin B₁₂ injections. Experimental parameters were optimized, including luminol concentration, urea-hydrogen peroxide (urea-H₂O₂) concentration, effect of pH, and sequence of addition of reactants for obtaining maximum CL, which was not explored previously. The limit of detection was 5 pg/ml, and the linear range was 10 pg/ml to 1 μg/ml with a regression coefficient of R² = 0.9998. The importance of these experimental parameters and the carbonate enhancement effect is discussed based on the knowledge of the mechanism of oxidation of luminol and decomposition of urea-H₂O₂ in the presence of vitamin B₁₂. Extraction of vitamin B₁₂ was carried out, and the observed recovery was 97-99.2% with a relative standard deviation in the range of 0.30-1.09%. The results obtained were compared with those of the flame atomic absorption spectrometry method.     

High-throughput chemiluminometric genotyping of single nucleotide polymorphisms of histamine, serotonin, and adrenergic receptor genes

Several pharmacogenetic studies are focused on the investigation of the relation between the efficacy of various antipsychotic agents (e.g., clozapine) and the genetic profile of the patient with an emphasis on genes that code for neurotransmitter receptors such as histamine, serotonin, and adrenergic receptors. We report a high-throughput method for genotyping of single nucleotide polymorphisms (SNPs) within the genes of histamine H2 receptor (HRH2), serotonin receptor (HTR2A1 and HTR2A2), and β₃ adrenergic receptor (ADRB3). The method combines the high specificity of allele discrimination by oligonucleotide ligation reaction (OLR) and the superior sensitivity and simplicity of chemiluminometric detection in a microtiter well assay configuration. The genomic region that spans the locus of interest is first amplified by polymerase chain reaction (PCR). Subsequently, an oligonucleotide ligation reaction is performed using a biotinylated common probe and two allele-specific probes that are labeled at the 3′ end with digoxigenin and fluorescein. The ligation products are immobilized in polystyrene wells via biotin-streptavidin interaction, and the hybrids are denatured. Detection is accomplished by the addition of alkaline phosphatase-conjugated anti-digoxigenin or anti-fluorescein antibodies in combination with a chemiluminogenic substrate. The ratio of the luminescence signals obtained from digoxigenin and fluorescein indicates the genotype of the sample. The method was applied successfully to the genotyping of 23 blood samples for all four SNPs. The results were in concordance with both PCR-restriction fragment length polymorphism analysis and sequencing.     

Enhanced chemiluminescence: A sensitive analytical system for detection of sweet potato peroxidase

3-(10'-Phenothiazinyl)propane-1-sulfonate (SPTZ) was shown to be a potent enhancer of anionic sweet potato peroxidase (aSPP)-induced chemiluminescence. The optimal conditions for aSPP-catalyzed oxidation of luminol were investigated by varying the concentrations of luminol, hydrogen peroxide, Tris, and SPTZ as well as the pH values of the reaction mixture. Addition of 4-morpholinopyridine (MORP) to the reaction mixture markedly increased the light intensity. Using SPTZ and MORP together enhanced the effect 265 times. The lower detection limit (LDL) of SPP was 0.09 pM, approximately in 10 times lower than that for the cationic isozyme c of horseradish peroxidase/4-iodophenol system. It was shown that aSPP in the presence of SPTZ produced a longer lasting chemiluminescent signal.