摘要:

AbstractWith the advent of enzymatically induced chemiluminescence and improved instrumentation for luminometry, ultrasensitive detection of a wide variety of analytes is now possible using standard immunoassay and DNA probe formats. Model molecular orbital calculations and literature precedent suggest that the singlet efficiencies observed upon decomposition of dioxetanes appended with donor substituted aromatic moieties are dependent on substitution pattern. We have recently discovered, in a series of 3‐(2′‐spiroadamantane)‐4‐methoxy‐4‐acetoxynaphth‐2′‐yl‐1,2‐dioxetanes, that enzymatic generation of a nonconjugated, charge transfer excited state results in luminescence of markedly different properties than that observed from an isomeric, conjugated excited state. An example of the former type, 3‐(2′‐spiroadamantane)‐4‐methoxy‐4‐(7″‐acetoxy)naphth‐2′‐yl‐1,2‐dioxetane (1) emitting at 550 nm, not only provides an increase in ΦCLbut exhibits a dramatic bathochromic shift of 80–110 nm from the 460 nm emission of the conjugated isomer 3‐(2′‐spiroadamantane)‐4‐methoxy‐4‐(6″‐acetoxy)naphth‐2′‐yl‐1,2‐dioxetane (2).These developments, along with the attendant glow‐type luminescence kinetics displayed during the enzymatic decomposition of the new ‘odd‐pattern’ dioxetane, allow the design o

ISSN:0884-3996    

DOI:10.1002/bio.1170050102

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractWe have used isoluminol steroid hormone conjugates in competitive heterogeneous chemiluminescence immunoassays (ClA) for estradiol, progesterone and estriol in body fluids. The assays did not require prior extraction of the steroid hormone with an organic solvent. Polyclonal or monoclonal antibodies, covalently coupled to polyacrylamide microspheres or adsorbed onto the plastic surface of microtritation plates via an intermediate second antibody, were used as solid‐phase. The latter solid‐phase system allows rapid processing of incubation mixtures. Sensitivie and reliable direct CIAs for estradiol in serum, progesterone in serum and in saliva and for estriol in saliva have been developed and their clinical utility has been assessed. Sensitivities ranged from 0.54 to 0.04nmol/l, and precision (% CV) ranged from 10% to

ISSN:0884-3996    

DOI:10.1002/bio.1170050103

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractA chemiluminescent assay for hepatitis‐B surface antigen is described which used an isoluminol derivative as the label. The assay is precise intra‐assay CV, 1.96‐2.45%; inter‐assay CV, 5.26‐8.11% and has a lower detection limit for hepatitis‐B surface antige

ISSN:0884-3996    

DOI:10.1002/bio.1170050104

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractBis(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 (

ISSN:0884-3996    

DOI:10.1002/bio.1170050105

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractPholasin is the photoprotein extracted from the marine bivalvePholas dactylus. It undergoes an oxidative chemiluminescent reaction to oxypholasin with superoxide anion, hypochlorite, peroxidases and other oxidants. Since the observed absorbance and chemiluminescent emission spectra of pholasin solutions cannot be brought about solely by the amino acids composing the protein, there has to be a chemiluminescent chromophore. However, little is known about the chemical nature of this molecule. This work seeks to identify the chemical structure of the luminescent prosthetic group of pholasin.Pholasin could not be reactivated using chromophores from the hydroidObelia geniculata(coelenterazine) and from the ostracod shrimpVargula(formerlyCypridina)hilgendorfi. Furthermore, the reaction product of theVargulachromophore could not be detected in solutions containing oxypholasin. Fluorescence analysis of such a solution revealed a compound with an emission spectrum (γmax480 nm; excitation at 320 nm), resembling the emission spectrum of the chemiluminescent reaction. This fluorescent substance was separated by gel filtration. It exhibited an apparent molecular mass of<2000. Fluorescence masurements of extracts of partially purified pholasin suggested that a flavin moiety may be involved in pholasin luminescence

ISSN:0884-3996    

DOI:10.1002/bio.1170050106

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractA severe dysfunction in the cellular response of human polymorphonuclear leukocytes (PMNL) to non‐opsonized zymosan was observed under a deficiency of extracellular Mg2+. The phagocytosis‐association native (luminol‐independent) luminescence (NL), as well as luminol‐dependent luminescence (LDL) (detected simultaneously and discriminated by spectral methods), was strongely inhibited. Apart from a general decrease of total light production, a Mg2+‐concentration‐dependent delay of the maximum of NL and LDL was observed. A disorder in recruitment of activated membrane‐bound NADPH‐oxidase of PMNL is suggested. The presence of extracellular Ca2+did not compensate for the Mg2+deficit. In the presence of Mg2+only a slight Ca2+‐dependent reduction of NL was obtained, but Ca2+seemed to selectively promote LDL. This may indicate a positive influence of Ca2+on the myeloperoxidase release from the cells. Experiments with the metalions‐chelating agents EDTA and EGTA, which complex Mg2+to differing extents, confirmed the important role of Mg2+in PMNL‐activation by

ISSN:0884-3996    

DOI:10.1002/bio.1170050107

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractThe characteristics of the luminol‐amplified chemiluminescence (CL) induced in mononuclear phagocytes interacting with PMA, FMLP or ionomycin were determined. Azide reduced the CL activity by more than 80%, while superoxide dismutase and catalase had minor effect on the monocyte CL response. The sensitivity of the monocyte CL response, to the addition of extra peroxidase differed depending on the stimulus used. Furthermore, no direct correlation was obtained between the CL response and superoxide anion or hydrogen peroxide production. In comparison with the response in granulocytes, minor quantitative differences were observed. The mechanism for the light‐generating reaction, seems to be the same in both cell ty

ISSN:0884-3996    

DOI:10.1002/bio.1170050108

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractThe incubation of macropages (MΦ) in the presence of lipopolysaccharides (LPS) usually results in the release of a variety of immunoregulatory cytokines such as interleukins (IL), tumour necrosis factor (TNF) and colony stimulating factors (CSF). We recently observed that conditioned media (CM) from LPS‐treated murine MΦ lines probably contain another protein endowed with granulocyte stimulatory activity. This cytokine, which has an apparent MW of about 55 kDa enhances the PMA‐induced luminescence of granulocytes and also stimulates their degranulation as measured by lactoferrin release. In contrast to IL1and IL6this factor is destroyed by brief treatment at pH 2, but is stable for 60 minutes at 65°C. Unlike CSF, its activity is unchanged by reducing agents such as beta‐mercaptoethanol. Furthermore, pretreatment of the MΦ with dexamethasone, in order to reduce the release of IL1and TNF, hardly reduces the effect on granulocyte activation. Finally, treatment with a neutralizing polyclonal anti‐murine TNF antiserum only partly abolishes its activity.These results show that, in addition to the already well‐described cytokines, LPS‐treated murine MΦ lines most probably secrete another granu

ISSN:0884-3996    

DOI:10.1002/bio.1170050109

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractA solid phase immunoassay for aldosterone using enhanced chemiluminescent detection has been developed. Monoclonal antibodies against aldosterone were used for the immune reaction and compared with polyclonal antibodies. Uniform Protein A coated polystyrene tubes were used as solid phase for the monoclonal antibody and second (anti‐rabbit) antibody coated tubes for the polyclonal antibody. Horseradish peroxidase was covalently linked to aldosterone as enzyme label. Optimum conditions were established for the generation and measurement of the luminescent reactions using luminol, p‐iodophenol as enhancer and hydrogen peroxide.The advantages of this assay are the high sensitivity with a detection limit of 100fg/tube, the prolonged luminescence signal with a simplification of the measurement (simpler detectors, external start pipetting) and the short measure time with the possibility of repeated measurement. The coefficients of variation were 4.2%–7.3% in the concentration range 140–1180 pmol/l. The assay showed a significant correlation (r= 0.91) with the ELISA.The aldosterone concentrations in plasma and saliva of patients with Conn's syndrome were significantly increased, and in patients with Addison's disease were found near the detectio

ISSN:0884-3996    

DOI:10.1002/bio.1170050110

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractThe mechanism of luminol chemiluminescence is a special case of nucleophilic addition to carbonyl compounds. The breakdown of the key intermediate, an alpha hydroxy hydroperoxide, produces a peracid ortho to an acyl diazene group. After intramolecular addition of the peracid, the energy from nitrogen expulsion is utilized in the formation of an anti‐aromatic endoperoxide. Rupture along the O,O bond leaves a substantial part of the ensuing phthalate in its excited state. The emitter is shown to be a mono‐protonated phthalate unaccessible by photoexcitation. The dark reaction is a concerted decomposion of the alpha hydroxy hydroperodixe to yield ground‐state phth

ISSN:0884-3996    

DOI:10.1002/bio.1170050111

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY