Ziva®-CPA
Principles of Ziva® Assays
Ziva®-CPA is an ELISA based ultrasensitive BrdU Cell Proliferation assay which uses a chemiluminescent substrate to detect the presence of an anti-BrdU antibody labeled with alkaline phospatase. The Ziva®-CPA assay has two different formats, the STANDARD and the FLEX formats. These formats differ in how the cells are prepared for the Ziva®-CPA assay. The assay procedure for both formats is the same from the stringency step forward.
An Introduction to Cell Proliferation Assays
Cell proliferation assays are used in a variety of important medical research applications. In its simplest form, cell proliferation can be detected and quantitated by counting labeled cells directly. Various cell staining techniques are used to facilitate cell counting. However, these direct methods are not useful for detecting the proliferation of small numbers of cells within a population of cells. Alternatively, cell proliferation can be indirectly detected and quantitated using assays such as MTT, XTT or WST-1 colorimetric assays, which detect the metabolic activity of a cell population as the cell number increases. A major shortcoming to these indirect methods has been high assay backgrounds and relative assay insensitivity. Another widely used indirect method for detecting cell proliferation is the chemiluminescent ATP detection assay. These indirect cell proliferation assays have very limited utility for detecting the proliferation of small numbers of proliferating cells within a population of many cells. This greatly limits the application of these indirect assays. As an example, for a T-cell proliferation assay, generally only a small fraction of a large population of viable cells present in the assay may be actively proliferating and of interest.
The "Gold Standard" method for detecting cell proliferation has long been the 3H-thymidine method, which incorporates tritiated thymidine into the synthesis of new DNA in proliferating cells. This method provides a method for detecting and quantitating a low number of proliferating cells, and is capable of detecting the proliferation of a small fraction of cells in a large population of cells. The Lower Limit of Detection (LLOD) of cells for the 3H-thymidine method is about 200-500 cells.
Current trends in lab techniques and requirements of smaller clinical laboratories have necessitated the use of non-isotopic labels and smaller numbers of cells. Current commercially available colorimetric, fluorescent, and chemiluminescent cell proliferation assays which detect newly synthesized cell DNA have been developed as an alternative to 3H-thymidine. These types of assays generally detect the incorporation of BromodeoxyUridine (BrdU) a DNA precursor into cell DNA by using a labeled anti-BrdU antibody and detection substrate, in an ELISA format. These non-isotopic BrdU ELISAs typically take an average of 2.3 hours to perform. However, most assays detection limits are in the 103 cell range, with very few of these assays claiming an assay cell LLOD equal to the 3H-thymidine method. Information is very limited with regard to these assays ability to detect a small fraction of proliferating cells within a large population of non-proliferating cells.
In contrast, Ziva® can be performed in less than 1 hour and is approximately 200-500 times more sensitive than the 3H-thymidine method (i.e., has a cell LLOD 200-500 times lower than the 3H-thymidine method). In a spike-in experiment, Ziva® has been used to detect 1-4 BrdU labeled actively proliferating cells in a background of 100,000 non-proliferating viable cells.