What is the Extraction Solution and when should I use it?
Several kits for measuring small peptides are supplied with an Extraction Solution (X123) that can be used to purify samples that would otherwise be unusable in those assays. This acidic organic solvent solution will precipitate proteins and other interfering substances, but leave the analyte of interest in solution. Any sample that might contain contaminants can be treated, but serum, plasma, and saliva must be extracted in order to get usable results from the Oxytocin (K048-H, K048-C), Arginine-Vasopressin (K049-C), or ET-1 (K045-H1) assays. To prepare samples, 1 volume of sample is mixed with 1.5 volumes of Extraction Reagent, vortexed, and then mixed for 90 minutes. After centrifuging the mixture for 20 minutes at 1660 g, the supernatant is removed to a separate tube and dried down in a SpeedVac. At this point, the sample can be reconstituted in the Assay Buffer supplied with the kit and measured in the assay. Please note samples can also be extracted with C18 solid phase columns if preferred; a protocol for that can be found in the resources section of our website at: www.ArborAssays.com/resources/#protocols.
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.
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.
What about a non-validated species?
Many analytes, such as steroids (Corticosterone etc.), cyclic nucleotides (cAMP and cGMP), or small lipids (PGE2, PGFM, etc) are exactly the same regardless of their source. Samples of these molecules from any species should therefore be 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 (K008-F1) or PKA (K027-H1) Activity Assays, or that detect the presence of an analyte without using antibodies, such as the Nitric Oxide (K023-H1) or Hemoglobin (K013-H1) 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”. Those kits that are limited to certain validated species will include that in their name, such as the Cystatin C Human Immunoassay kit (K012-H1).
What is matrix interference?
In addition to the analyte you are trying to measure, many samples contain additional elements, such as proteins and lipids, which can interfere with the free association of the target analyte and the specific antibody in the kit. In the standard curve wells, the pure analyte is simply dissolved in assay buffer, so those potentially interfering components are not present. When the assay is performed, you may encounter a situation where the sample well and a standard well contain exactly the same amount of analyte, but additional components in the sample well interfere with binding to the specific antibody. As a result these wells will generate different amounts of signal even though the amount of analyte is the same.
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.
Do I need to use dissociation reagent?
Dissociation reagent is provided in some steroid and other assays to minimize binding of some hormones to sample proteins. It only needs to be used on serum or plasma samples, as steroids in those samples are associated with carrier proteins and not available to be detected in the assay. Samples in matrices such as saliva, feces, and urine will not need dissociation reagent. Most assays for steroid analytes are supplied with a special Dissociation Reagent that should be used to treat serum and plasma samples. The reagent frees steroids for measurement from specialized binding proteins and other carriers such as albumin to which steroids will bind when in circulation. After treating serum and plasma samples with the Dissociation Reagent, samples must be further diluted with Assay Buffer before testing. This dilution is necessary to eliminate matrix interference from the dissociation reagent itself. Check the kit insert for the recommended dilution in each assay.
How do I prepare my samples for GSH measurement?
We get a number of questions about our very popular assays for measuring Glutathione (K006-F, K006-H). 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 (K006-F), reduced GSH and oxidized GSSG can be determined in the same well. In the Colorimetric kit (K006-H), 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.
What’s the advantage of CLIA vs EIA?
Chemiluminescent assays (CLIA) are usually more sensitive than colorimetric assays (EIA) for the same marker. For example, the cAMP EIA Assay (K019-H1/H5) has a sensitivity of 5 fmol, while the cAMP CLIA Assay (K019-C1/C5) is sensitive to 1 fmol. CLIA assays require a plate reader capable of measuring glow luminescence from the assay, different from the colorimetric readout of optical density.
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. Alternatively, free data analysis software for all of Arbor Assays’ kits can be found online at www.MyAssays.com.
Why do I have to Acetylate my Samples?
All the DetectX® kits for Cyclic Nucleotides (cAMP, K019-H and K019-C, and cGMP, K020-H and K019-C) are supplied with reagents that can be used to acetylate standards and samples. The specific antibodies in these kits are more reactive to nucleotides that have been acetylated, so treating samples this way will allow for a more sensitive measurement of cAMP or cGMP. There are a couple things to note about acetylating, however. First, the acetylating reagents will cause proteins and lipids in the samples to precipitate. Only samples that have been diluted to the concentration which will be used in the assay should be treated. Second, acetylation does add another step to the process. In general, we recommend testing samples initially without acetylating. The regular assays are very sensitive, and most samples can be analyzed without the additional step. If the concentration of your samples is too low, or if they need to be diluted very far to eliminate matrix interference, then acetylation can be a useful tool for getting good results from the assay.
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 those 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 limit of sensitivity that is a 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.
What are assay Sensitivity and Lower Limit of Detection (LLD)?
(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). LLD 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 LLD is higher than the sensitivity.