Q. Why do I have to use 4PL curve fitting?

The sigmoidal shape of the standard curve in competitive assays is most accurately fit by a four-parameter logistic model (4PLC). Alternate models such as linear, exponential, or log-log, give inaccurate readings, particularly at high and low concentrations. Most plate readers have the ability to fit data using 4PLC methods from the standard curve. Check with your plate reader’s manufacturer if you are unsure.

Q. How do I prepare my samples for GSH measurement?

We get a number of questions about our very popular assays for measuring Glutathione (ZX-44100, ZX-33100). In both of these assays, the detector used to quantitate GSH in samples will also react with any free thiol group present in the sample. For this reason, proteins or other potential interfering components must first be removed. This is accomplished by treating all samples with 5% 5-sulfo- salicylic acid dihydrate (SSA), which causes larger proteins to precipitate but leaves glutathione in solution. After centrifuging, the supernatant can be collected and diluted with Assay Buffer to a maximum of 1% SSA before testing in the assay. In the Fluorescent kit (ZX-33100-96, ZX-33100-480), reduced GSH and oxidized GSSG can be determined in the same well. In the Colorimetric kit (ZX-44100-96), treating samples and standards with 2-Vinylpyridine (2VP) — a chemical that prevents reduced glutathione (GSH) from reacting with the detector — allows for the measure of Oxidized Glutathione (GSSG) separate from any Reduced GSH present. To determine the concentration of free GSH, subtract the concentration of Oxidized GSH from the concentration of total GSH determined separately from the same samples not treated with 2VP.

Q. How do I determine the best dilution for samples?

First look up similar studies in PubMed and try to determine what normal levels are. We have optimized recommended dilutions for most validated sample types to minimize any sample interference in the result. If you are running a sample type we do not list please contact us first to see if we have any data on that type. If we do not you may have to run linearity and spiked sample dilution experiments to determine what dilution will give you linear results.

Q. What about a non-validated species?

usable in EIAs or CLIAs, even if they have not been specifically tested. In addition, kits that measure the activity of a sample, such as the Glutathione-S-Transferase (ZX-33103) Activity Assays, or that detect the presence of an analyte without using antibodies, such as the Nitric Oxide (ZX-44107) or Hemoglobin (ZX-44112) Detection Assays, are not limited in any way by a species- specific interaction and therefore will work with samples from any species. All kits that fall into these categories are indicated as being “Multi Species”.

Q. Can I use a non-validated sample type?

Most sample types not specifically tested during kit development can still work, but will require testing and optimization. Samples might need to be diluted or may have to be extracted to eliminate matrix interference. Optimal dilutions to get sample values to fall within the range of the standard curve will also have to be determined. In the end, if the analyte is still present in concentration high enough that it falls within the range of the standard curve after the sample has been diluted sufficiently to eliminate matrix interference, or the sample can be extracted and an acceptable amount of the analyte recovered, then the sample will work.

Q. In an EIA or CLIA, what are NSB, B0, and zero standard?

(a). NSB (non-specific binding) This represents signal from non-specifically bound peroxidase conjugate in a competitive immunoassay. This signal is generated from conjugate retained on the plastic itself, and the background signal from the substrate.
(b). B0 (binding for the zero standard, maximum binding well) this represents the maximum signal from enzyme captured by the specific antibody in competitive EIA or CLIA immunoassays. All other standards and samples are expressed as a percentage of this value.
(c). Zero Standard (background signal for sandwich assay) in an immunometric assay this represents the minimum signal from the assay. The Zero Standard becomes part of your standard curve..

Q. Can I change the standard range?

All assays have been tested and designed to give accurate and reproducible results over the range of standards shown in the insert. Additional points higher or lower than described concentration may not improve assay performance. Changed standard concentrations could cause inaccuracies by not giving adequate data at important assay points. Remember also that assays have a sensitivity limit which based on the function of the antibodies and buffers used in the test. Simply adding additional standard concentrations to the lower end of the curve will not make the test more sensitive. Following the standard curve dilution recommendations will generally give you the best results.

Q. What are assay Sensitivity and Lower Detection Limit?

(a). Sensitivity is the lowest value of analyte in assay buffer that the assay can statistically differentiate from background. It is a calculated value, determined by comparing signal from many replicates of low standard wells and zeros. It is possible for the assay’s sensitivity to be higher than the lowest standard point. Sample values found to be below the assay’s sensitivity should be considered to be too low to detect.
(b). Detection Limit is similar to sensitivity, but determined by testing a native sample. Replicate wells of dilute samples and zeros are compared, and the lowest concentration of sample that can be statistically differentiated from zero is determined. In most cases the Detection Limit is higher than the sensitivity.