LUMIstar Omega

SPARCL refers to an immunoassay that does not require washing steps. An analyte in a solution is detected by binding two antibodies: one is coupled to horse radish peroxidase (HRP), the other is coupled to the HRP substrate Acridan. In the presence of the analyte, binding of both antibodies brings the enzyme and substrate into proximity. The enzymatic reaction is started by the addition of H₂O₂, which immediately produces a light proportional to the amount of analyte. Thus, SPARCL assays require to be read at the same time as the injection of the trigger solution.

The CLARIOstar^® microplate reader, which is equipped with the dedicated H3 injection needle for simultaneous injection and reading, fulfils this task. The analyte IgG was detected down to a concentration of 45 ng/ml in 96 wells and down to 15.6 ng/ml in 384 well format. BMG LABTECH's MARS analysis software facilitates subsequent calculation of analyte concentration.

Multiplexing AlphaLISA^® type assays will increase throughput while sample expenditure and costs are minimized. The ability of the PHERAstar^® FS microplate reader to perform simultaneous dual emission detection of this assay will allow users to double the information obtained from each well in the same amount of time as needed for a single emission assay.
 

Antibody pairs were selected for each of two protein targets. For each protein target, one of the paired antibodies was conjugated to either AlphaPLEX™ 545 nm acceptor beads or AlphaLISA^® acceptor beads. The other antibody of each pair was biotinylated. A mixture containing acceptor beads and biotinylated antibodies for both target proteins was prepared and added to the reaction plates.
 

After laser excitation two different emission wavelengths were measured using the AlphaLISA SDE optic module. Excellent assay parameter for S/B as well as for Z' indicate the microplate readers capability to measure Alpha signals simultaneously without compromising data quality.

Detection of biomarker interleukin 8 (IL-8) is a good measure to evaluate the differences of normal versus pathogenic processes in the human body. Here we will show how the SPARCL technology can be used in combination with the BMG LABTECH microplate reader instrumentation to detect IL-8 as well as IgG.
 

The SPARCL technology is based on a specific antibody/antigen interaction that brings acridan and HRP into close proximity. After addition of a hydrogen peroxide trigger solution a flash of light follows. The luminescent flash exists only for a very limited time. A typical SPARCL pattern of flash luminescence shows that most of the signal is already gone after 1 second. Because of this situation it is necessary to use an instrument that is able to inject trigger solution and measure flash luminescence at the same time.

© 2015 Lumigen, Inc. All rights reserved. Used with permission. Lumigen and SPARCL are trademarks of Lumigen, Inc. and are registered in the USPTO. Lumigen is a Beckman Coulter company.

Mycoplasma are small-sized bacteria that are well-known as contaminant of laboratory cell cultures. Without having a cell wall around their membrane they cannot be targeted with antibiotics. That makes them a big problem and often the whole culture is lost.
 

As mycoplasma can influence experimental results obtained for a certain cell line, cell culture labs need to check regularly if their cultures are contaminated by mycoplasma species. Here we show the application of the MycoAlert™ assay from Lonza to perform this test. The assay is specific for mycoplasmal enzymes that are not present in eukaryotic cells. After addition of substrate a high luminescent signal is created only in viable mycoplasma.
 

The luminescence detection was done in a CLARIOstar^® microplate reader. The instrument comes with the MARS Data Analysis software that allows the user to do calculations and validations to quickly identify samples which may be contaminated.

Use of primary cells is preferred in drug research as they lack immortalizing mutations and are not monoclonal. Therefore, primary cells predict drug responses more reliably. Upon activation of the drug relevant G-protein coupled receptors, second messengers become active such as calcium ions or cAMP. Two assays that report on these second messengers and are amenable to primary cells are presented here.

The cytosomal release of Ca²⁺ was measured in 96 well plates with the Clonetics^TM calcium sensor that was transiently transfected into human umbilical vein endothelial cells (HUVECs). The cells were stimulated by injection of histamine. The GloSensor cAMP sensor was transiently transfected into human mesenchymal stem cells to display cAMP generation upon varying doses of Formeterol, PGE1 or Forskolin. The cAMP assay was performed in 96- and 384-well plates.

The luminescence plate readers by BMG LABTECH reliably detected the fast changes in Ca²⁺ and determined the EC₅₀ values of cAMP-stimulating agents in primary cells.