MycoAlert® Mycoplasma Detection Assay From Lonza

Screening for the presence of mycoplasma in cell lines is an important task in any laboratory that practices tissue culture, as contamination of cell lines can cost money and time if not detected early, especially as cells do not show visual effects of mycoplasma contamination until late in infection. Traditional methods of detecting mycoplasma include PCR, immunofluorescence and isolation which require a reasonable amount of time to perform. The MycoAlert® Mycoplasma Detection Assay from Lonza has been designed as a replacement method to screen for mycoplasma contamination. The assay utilizes a bioluminescent reporter assay and can be performed in either a single sample or 96-well format.

The kit includes lyophilized samples of MycoAlert® Reagent and Substrate and a bottle of MycoAlert® Assay Buffer. Different sizes are available of 10, 25, 50 and 100 tests each. An additional positive control kit of 10 positive tests is available separately (does not contain mycoplasma). The presence of mycoplasma is determined in a two step process. The Reagent contains a proprietary substrate that is catalyzed by mycoplasma-specific enzymes present in media from a cell culture, resulting in the conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP). The ATP levels are quantified by addition of the Substrate which contains luciferin and luciferase. The luciferase catalyses a light-producing reaction between luciferin, ATP and oxygen. The intensity of the light emitted is quantified using a luminometer, and is directly related to the ATP in the sample. By comparing the intensity before and after addition of the Substrate, it is possible to tell whether the cells are positive or negative for mycoplasma.

The protocol for testing is straightforward and multiple samples can be processed at one time. All reagents are brought to room temperature beforehand to ensure readings are accurate, and gloves are essential to prevent ATP from your skin contaminating samples. Working solutions are prepared by addition of Buffer to both Reagent and Substrate. A 2 ml media sample from the cells is collected from cells that have been cultured for several days. This sample is centrifuged for 5 minutes to remove cell debris, then a 100 ìl aliquot is placed in a 96-well plate or cuvette. To begin the assay, 100 ìl of MycoAlert® Reagent is added, and samples are incubated for 5 minutes. A reading of the cells is taken with the luminometer (Reading A) and then 100 ìl of the MycoAlert® Substrate is added. After a10 minute incubation, the luminescence is determined again (Reading B). A ratio is then calculated (Reading A/Reading B). A value below 1 is considered negative, slightly above 1 is borderline (and requires retesting) and anything higher is positive.

I have used this kit for over a year, and have found it indispensable for routine screening of tissue culture, with a single sample taking only 20 minutes to analyze once all reagents are at room temperature. It is expensive per test but the speed and convenience with which you can perform the assays compared to traditional methods makes this acceptable.