Help/FA&Q
1. Can a partial Youkelife ELISA plate be used?
The Youkelife ELISA plates have removable strips. Unused strips may be removed from the plate, returned to the foil pouch containing the desiccant pack, and stored at 2-8° C for up to one month.
2. Can distilled water be used instead of deionized water to dilute kit diluents?
Both deionized and distilled water can be used to dilute the dilution concentrate.
3. Can ELISA Kits be used with tissue homogenates (or other non-validated sample types)?
ELISA kits can detect them and can be consulted with Youkelife Technical Support for optimal results.
4. Can ELISA plates be stacked on top of each other during assay incubations?
To ensure consistent environmental conditions for all plates, it is recommended that ELISA plates are not stacked during incubations.
5. Can the standard curve be extended in either direction?
Youkelife cannot support standard concentrations outside the validated standard curve. The standard curve range has been validated for reliable and accurate performance across multiple kit lots and operators. The lowest standard concentration is the lowest limit that Youkelife can guarantee for reliable assay measurements.
6. Does Youkelife provide ELISA testing recommendations for inactivating samples thought to contain Covid-19 or other viruses?
Youkelife does not provide recommendations for inactivation of viruses in samples. Heat and chemical pre-treatments may cause conformational changes to most proteins. These proteins do not revert to their normal conformation without the help of molecular chaperones.
7. Does Youkelife add Heterophilic Blocking Reagents to Assay diluents and Calibrator Diluent provided in ELISA and immunoassay kits? Will you recommend treating samples with HBT reagents?
Youkelife does not add HBT reagents to diluents, but instead formulates specialized diluents that are designed to alleviate false positives to achieve the most accurate results. Our diluents are designed to minimize interfering factors such as binding partners, soluble receptors, HAMA, and rheumatoid factor. With Youkelife immunoassays, it is not necessary to add heterophilic blocking reagents to the samples.
8. Have Youkelife ELISA kits been evaluated for cross-reactivity with different species?
When available, recombinant proteins for different species are evaluated in an ELISA kit for cross-reactivity and interference.
9. What is the reason for the large coefficient of variation of replicating wells in the same sample ?
The two main reasons for high variability in an assay is related to pipetting & wash technique.
10. I used your recombinant protein as a control in the corresponding ELISA kit. Why am I seeing discrepancy in mass values?
First, a large dilution is required to place the recombinant protein on the standard curve range. Typically this is a dilution from μg/mL to pg/mL. Any dilution step can introduce inaccuracy. Any pipetting error or mis-calibrated pipet can result in apparent over- or under-recovery. Second, this Standard is calibrated to standards established when the kit was initially developed. The protein determination of these initial standards became the Master Calibrators to which all new standards are formulated. This ensures Youkelife immunoassay kits with consistency between manufacturing lots. In general, the recovery rate of the prescribed amount on the Quality Control vial is +/- 15% when using the Youkelife ELISA to determine a protein concentration. There may be slight differences in the immunologically recognizable mass between lots of Contorls, so the Standard concentration provided on the Quality Control vial may vary from lot-to-lot when measured in the ELISA. If you are using proteins to make controls, it is better to calculate the concentration value based on the measurements in the ELISA and not use the mass on the vial when setting control levels.
11. What are the dimensions of the ELISA microplates included in Youkelife ELISA kits?
The plates included in Youkelife ELISA kits meet standard footprint dimensions for microplates, 200 mm x 150 mm x 58 mm (length x width x height).
12. What is a competitive ELISA?
In the competitive immunoassay approach, also termed "labeled analyte technique", there exists a competition between the endogenous unlabeled antigen and an exogenous labeled antigen for a limited amount of antibody binding sites. Therefore, a decreasing signal indicates higher concentrations of the analyte being measured.
13. What is a sandwich ELISA?
A sandwich ELISA uses an immobilized capture antibody specific for the analyte of interest in a sample. After the analyte is bound to the immobilized antibody, a labeled secondary antibody specific for the analyte is used for detection. The analyte is "sandwiched" between the two antibodies. The sandwich ELISA is extremely sensitive, and the values obtained are quantitative when compared with a standard curve.
14. What is the expected range of OD values for the highest standard concentration?
Many factors can influence the high standard OD value. Signal can vary according to operator technique for the standard curve preparation, washing method, incubation variability, and plate reader calibration. The maximum assay signal is expected to measure between 1.0 and 3.5 OD.
15. What is the half life of a cytokine in serum/plasma/CCM for ELISA?
Youkelife does not determine the half life of cytokines in natural samples such as serum, plasma, or cell culture supernates. Our general recommendation is to assay the sample immediately or aliquot into single use volumes and store these samples frozen. We recommend avoiding repeat freeze-thaw with these samples to avoid protein degradation.
16. What is the purpose of wavelength correction? Do I need to use 570 or 630 nm?
The purpose of wavelength correction is to correct for optical imperfections in the plate. Youkelife generally recommends in ELISA protocols to set wavelength correction to 630 nm. Wavelength between 540 - 690 nm can be used as correction. Readings made directly at 450 nm for ELISA assays may be higher and less accurate.
17. Why are my wells green after adding the Stop Solution?
This happens when the substrate in the well does not completely mix with the stop solution. After addition of the stop solution, tap the plate gently or place on a shaker until the mixture in the wells turns yellow. To prevent this from happening, we recommend touching the pipette tip to the side of the well at a 45 degree angle to the plate and quickly dispensing into the well. This angle and speed helps disperse the Stop solution throughout the well.
18. Why doesn't the assay range extend to the stated sensitivity?
Sensitivity is the lowest measurable value that is statistically not equal to zero. It is calculated based on the signal of the background and the inherent variability of the assay. It is commonly determined by taking the mean O.D. plus two standard deviations from 20 zero replicates. This value can be obtained from standard curve regression fitting.
19. Why is there a sample dilution necessary in this ELISA Kit?
There are primarily two reasons for dilutions. In some assays most samples read above the standard curve, thus requiring a dilution for analyte levels to fall within the range of the assay. A second reason for dilution is to limit interference due to factors in complex matrices.
20. Why must I use polypropylene tubes for standard dilutions in certain assays?
Certain proteins or analytes will bind to glass and polystyrene, but do not readily bind to the polypropylene tubes.