The use of a CCD imaging luminometer in the quantitation of luminogenic immunoassays

We describe the application of a CCD Imaging Luminometer (CCDIL) for the detection and quantitation of rapid, simultaneous, peroxidase-linked chemiluminescent immunoassays of multiple samples of human serum alphafetoprotein (AFP). Results from some analogous immunoassays (total IgE, and thyroid stimulating hormone (TSH)) are included for comparison. Values for precision and antigen concentration obtained using the CCDIL and colorimetric versions of the immunoassays, on human serum samples, were in good agreement. The flexibility of the CCDIL is demonstrated; its ability to detect and quantitate antigen (particularly AFP) on a variety of solid phases is indicated. The work on the AFP immunoassays illustrates not only the flexibility of the CCDIL for sample presentation on a variety of solid phase systems, but also some relative merits of such systems.     

Studies on PVP hydrogel-supported luminol chemiluminescence: 2. Luminometer calibration and potential analytical applications.

The use of poly(N-vinyl-2-pyrrolidone) (PVP) hydrogel-supported luminol chemiluminescence (CL) for the automatic determination of hydrogen peroxide and the quantification of the antiradical capacity of Trolox is described. The hydrogel containing luminol and hemin is prepared directly on a 96-well microplate and can be stored for up to 3 months without significant decrease in CL quantum yields. Furthermore, this system can also be used as a secondary light standard for the calibration of microplate luminometers.     

Opsonophagocytosis of bacteria studied by chemiluminescence in microtitre plates

A protocol for polymorphonuclear leukocyte chemiluminescence (PMN CL) assays of opsonophagocytosis was developed for a microtitre-plate luminometer. The complete procedure was performed in a single microtitre plate and was simpler and more efficient than previous protocols. The kinetics of the PMN CL response were best when microtitre plates were incubated on a shaking incubator between readings. The new protocol was used in a study of the pathogenicity of Corynebacterium jeikeium, an organism found in association with infection in the immunocompromised. No differences were found when PMN CL induction by 15 strains of C. jeikeium were compared with 15 isolates of other corynebacteria. Both groups of organisms required complement for efficient opsonophagocytosis; C. jeikeium strains showed no requirement for specific antibody. Resistance to opsonophagocytosis does not appear to be an explanation for the increased pathogenicity of C. jeikeium. Microtitre-plate luminometers are particularly well suited to bacterial opsonization studies where large numbers of strains often need to be assessed.     

A solid-state,portable instrument for measurement of chlorophyll luminescence induction in plants

A newly developed compact instrument is described for the measurement of chlorophyll luminescence induction in plants. The instrument operates with a pulsed light emitting diode (LED) as light source and a photodiode as luminescence detector. A special emitter-detector geometry provides for high irradiance of the sample and efficient collection of luminescence by the detector. With insertion of appropriate filters the same probe is also suited for measuring prompt chlorophyll fluorescence. The instrument shows considerable flexibility with respect to pulse frequency, relative lengths of light/dark intervals and luminescence sampling periods. Due to a selective amplifier system only that part of luminescence is processed which is induced by the individual excitation pulses. By this approach, the problem of slow phase accumulation, encountered with conventional phosphoroscopes, is eliminated. Some examples are given for system operation, demonstrating satisfactory performance in measurements with intact leaves and isolated chloroplasts.     

A comparison of whole blood neutrophil chemiluminescence measured with cuvette and microtitre plate luminometers

Results obtained by measuring human whole blood neutrophil chemiluminescence (CL) using the BioOrbit 1251 cuvette luminometer and the Immunotech LM‐01T microtitre plate luminometer are compared in this study. Opsonized zymosan, phorbol myristate acetate, N‐formyl–Met–Leu–Phe and calcium ionophore A23187 were used as activators. The CL response of neutrophils to their stimulation with the individual types of activators tested was fully detectable using either type of the luminometers. The kinetic curves of CL activity obtained from both the cuvette and the microtitre plate luminometers had similar characteristics. The only insignificant difference observed when comparing the kinetic curves was in the rates of the CL reactions. The peak CL response of activated neutrophils was reached faster when using the luminometer BioOrbit 1251 than with the luminometer Immunotech LM‐01T. A likely reason for this difference is the mode of transporting samples during the measurement, inducing different degrees of agitation. However, although this fact needs to be considered when interpreting results, both types of luminometer can be fully utilized in both research and clinical laboratories. Copyright © 2002 John Wiley & Sons, Ltd.     

A review of bioluminescent ATP techniques in rapid microbiology

Use of firefly luciferase to assay adenosine triphosphate (ATP) extracted from microorganisms provides an easy means to enumerate microbes within minutes. The small amount of light produced is proportional to ATP and thus microbial number. The average bacterium contains around 10^?15 g ATP per cell. Present reagents permit detection of 10³ cells per tube. Luminometers currently on the market detect about 10^?12 g ATP. Proper extraction of ATP from the microbes is an essential part of any protocol, as is the removal of non-microbial ATP from, for example, somatic cells also present in samples. The technique may be applied to a wide range of samples, for example food and beverages and clinical samples such as urine. The ATP assay gives a global measure of microbial numbers, i.e. it is not species specific unless a species separation step is included in the protocol.     

Attomol‐level ATP bioluminometer for detecting single bacterium

We have developed an automated high‐sensitive ATP bioluminometer for detecting single bacterium. The apparatus consists of a tube rack for setting reagents and samples, two washing baths for preventing sample carry‐over from dispenser nozzle, and x‐, y‐, z‐ actuators for moving the dispenser, and an high‐sensitive optical system. The reaction tube was selected to reduce the background signal intensities for the ATP bioluminescence measurement. The background signal intensity of the reaction tube was 18 RLU, which is almost the same as the dark counts of the photomultiplier (16 RLU). The ATP calibration curve was linear from 0 to 5 amol (its slope = 22.4 RLU/amol and 3.3 SD of the blank sample signal = 17.9 RLU), and the detection limit of 0.8 amol was obtained. The relationship between intracellular ATP and CFU in Escherichia coli (ATCC25922) was kept linearity from 0 to 20 CFU, and the intracellular ATP (amol) per CFU was calculated to be 3.3 amol/CFU (R2 = 0.9713). Moreover, the relationship between intracellular ATP and CFU in Staphylococcus aureus (ATCC25923) was also kept linearity from 0 to 30 CFU, and the amol/CFU was calculated to be 1.6 amol/CFU (R2 = 0.9847). The automated ATP bioluminometer has ultra‐high sensitivity and will be a powerful tool for measuring ATP luminescence derived from small number of bacteria.     

Luminometer development in the last four decades: recollections of two entrepreneurs

This article, written by two entrepreneurs in luminescence, traces their involvement in the major part of the interconnected innovation and development of luminometers, adenosine triphosphate (ATP) bioluminescence and other technologies from the mid‐1970s to 2011 that ushered in much of the field of luminometry as we know it today. Key developments leading to current commercial applications of ATP bioluminescence, luminescence immunoassay, cellular luminescence, reporter gene and other applications are described from the first tube luminometers derived from early luminescence studies using liquid scintillation counting technology to measuring bioluminescence from crude ATP and firefly tail extracts. Copyright © 2012 John Wiley & Sons, Ltd.