ELISA Frequently Asked Questions
- Platinum ELISA Kits
- Instant ELISA® Kits
- High Sensitivity ELISA Kits
- Ready-SET-Go!® ELISA Kits
- Antibodies for ELISA General Information
- Cytokine ELISPOT Product Line: Antibody Pairs and Ready-SET-Go!®
- FlowCytomix™ Multiple Analyte Detection System
Platinum ELISA Kits
Q. What do Platinum ELISA Kits contain?
All reagents necessary to perform 1 or 10 plate ELISA assays, including optimized capture-antibody pre-coated plates.
Q. Which samples are suitable for Platinum ELISA Kits?
Most ELISAs are suitable for serum, plasma, cell culture supernatants and other body fluids. Detailed information is given in the product manual for each individual kit.
Q. Do I have to run the Platinum ELISA standards and samples in duplicates?
No, but it is highly recommended to make at least double determinations of each sample and standard point.
Q. Is it possible to use the reagents of other Platinum ELISA kits/lots?
The reagents within the Platinum ELISA kits are not interchangeable between different lots of the same kit or between kits against different analytes.
Q. Can I always use the same manual for different ELISA kit lot numbers?
We recommend that you use the manual included with the Platinum ELISA kit.
Q. Is it necessary to wash the Platinum ELISA plates before usage?
An initial washing step is not necessary but recommended as accuracy may be increased.
Q. Are shorter or longer incubation times possible?
Incubation times should be followed exactly as stated in the product manual to ensure optimal Platinum ELISA test performance.
Q. Can I omit the dilution of my Platinum ELISA samples with assay buffer/sample diluent when performing the assay?
We recommend following the instructions for sample dilutions as provided in the manual. We cannot guarantee optimal performance of the assay if internal assay dilutions as instructed were omitted.
Q. What if it does not look like the supplied reagent volume is sufficient for running the Platinum ELISA?
In case of small reagent volumes, please spin down vial before use to make sure to all contents are collected in the bottom of the tube.
Q. Is it possible to store the reagents other than indicated?
Performance of Platinum ELISA cannot be guaranteed if reagents are not stored as indicated.
Q. Do you have information regarding the normal values detectable with your Platinum ELISA Kit?
You can find expected "normal levels" listed in the Platinum ELISA Kit manual under "Expected Values". Please be aware that these normal values were obtained in our laboratories with a limited number of samples. We strongly suggest that each laboratory perform studies in order to establish normal range for their sample populations.
Instant ELISA® Kits
Q. How many tests can I perform with an Instant ELISA Kit?
With an Instant ELISA, 128 tests (includes samples plus standards) can be performed. The number of tests results from the following composition:
- One 96 well plate designed only for samples; 96 samples
- Two 8-point standard curve in duplicates; 32 wells; Thus the kit can be used at two different time points, each with their own standard curve.
Q. Do I have to add recombinant standard to the wells?
No, the recombinant standard is included as a component of the lyophilized reagents in the wells.
Q. Do I have to add the secondary antibody to the wells?
No, the secondary antibody is included as a component of the lyophilized reagents in the wells.
Q. Do I have to dilute my samples before adding them to the wells?
In general, one can use undiluted samples with the Instant ELISA. Please refer to the product manual to determine if dilution of the sample is required for your kit of interest. The volume of sample required for the individual kits will also be listed in the product manual.
Q. Is the reconstitution volume of the standards the same for different lot numbers?
For some Instant ELISA kits, the reconstitution volume of the standard and blank wells may differ (these components are lot specific). Please use the manual provided in the Kit for specific volumes to use.
Q. How do I calculate my samples?
The calculation factor is always stated in the manual.
High Sensitivity ELISA Kits
Q. Can the amplification reagents be prepared ahead of time?
No, the amplification reagents have to be prepared immediately before use. The reagent AR1 contains methanol which evaporates easily.
Q. Can I deviate from the recommended incubation times?
No, after adding the amplification reagents to the sample wells, the incubation times have to be followed exactly for optimal use.
Q. Can I perform the incubations at higher temperatures?
Incubations should occur at ambient temperatures not higher than 25°C.
Q. Are the wash steps necessary?
Yes, please follow the washing steps exactly as outlined in the product manual (for optimal readings: an automatic plate washer is recommended).
Q. How should I prepare the standards for use with the High Sensitivity Kits?
We recommend that you prepare the standard in Eppendorf tubes.
Ready-SET-Go!® ELISA Sets General Information
Q. What do Ready-SET-Go! ELISA Sets contain?
- Capture Antibody: Pre-titrated, purified antibody
- Detection Antibody: Pre-titrated, biotin-conjugated antibody
- Standard: Recombinant cytokine for generating standard curve and calibrating samples
- ELISA Coating Buffer Powder
- Assay Diluent: 5X concentrated
- Detection enzyme: pre-titrated Avidin-HRP
- Substrate Solution: Tetramethylbenzidine (TMB) Substrate Solution
- Certificate of Analysis: Lot-specific instructions for dilution of antibodies and standards
- 96 Well Plate: Corning Costar 9018 (included with product Cat. #’s ending in suffixes -22, -76, -86)
Q. What are the storage conditions for your Ready-Set-Go! ELISA Sets?
All of the reagents are to be stored at 4°C upon receipt, except for the recombinant standards. The recombinant standards MUST be stored at or below -80°C to ensure stability and performance.
Q. What is the expiration date for your Ready-Set-Go! ELISA Sets?
The majority of our Ready-Set-Go! ELISA Sets are guaranteed for one year from the date of receipt. However, we do have several sets that are guaranteed for 6 months upon date of receipt. Please refer to the Certificate of Analysis for shelf-life information.
Q. What is the sensitivity of your Ready-Set-Go! ELISA Sets?
The sensitivity ranges of our Ready-SET-Go! ELISA sets vary depending on the cytokine/protein being measured. Please refer to the product datasheet for set specific information.
Q. What concentrations should I use for my recombinant standard?
Each Ready-Set-Go! ELISA Set comes with its own Certificate of Analysis (CofA). The directions on the CofA should be strictly followed to ensure performance of the reagents. The CofA gives specific instructions on how to dilute each component of your lot specific set. The instructions on the CofA should not be deviated from in order to ensure accurate results.
Q. I want to collect my tissue culture supernatant daily for a time-course study and run all of my samples at once by ELISA. How should I store my supernatants?
The supernatants can be harvested and stored -20°C. Avoid freeze/thaw cycles. It is recommended to briefly centrifuge samples after collection to pellet any dead/floating cells remaining in the supernatant. Transfer to new tube for storage.
Antibodies for ELISA General Information
Q. Do you offer antibody pairs for use in an ELISA?
Please see our Antibody Pairs Chart for a complete listing of the capture and detection antibodies, and recombinant protein standards available.
Q. What other reagents will I need to run an ELISA using the antibody pairs?
eBioscience provides the following components needed to run a successful ELISA. They include the following:
- High-affinity binding NUNC Maxisorp plates
- Coating Buffer
- 5X Assay Diluent (cat. no. 00-4202)
- Avidin-HRP (cat. no. 18-4100)
- Super AquaBlue ELISA substrarate (cat. no. 00-4203) or TMB (cat. no. 00-4201)
For ELISA pairs, eBioscience recommends using the HRP compatible substrate, Super AquaBlue (read at 405nm). Our Ready-Set-Go! ELISA Sets include TMB substrate (read at 450nm).
Q. What are the differences between TMB and Super AquaBlue?
TMB is a faster developing, stronger substrate that yields greater amplification/sensitivity/background. The use of an acid stop solution produces a yellow end product read at 450nm. Super AquaBlue is a slower developing blue/green substrate particularly useful for kinetic studies.
Cytokine ELISPOT Product Line: Antibody Pairs & Ready-SET-Go!®
Q. What are the components of the Ready-Set-Go! ELISPOT Sets?
- Capture Antibody: Pre-titrated, Functional Grade (low endotoxin) purified antibody
- Detection Antibody: Pre-titrated, biotin-conjugated antibody
- ELISPOT Coating Buffer
- Assay Diluent: 5X concentrated
- Detection enzyme: pre-titrated Avidin-HRP
- Certificate of Analysis: Lot-specific instructions for dilution of antibodies and enzyme
Q. Do your antibody pairs work in ELISPOT as well?
Most, but not all, of our ELISA antibody pairs work for ELISPOT. We continue to evaluate and optimize our anti-human and anti-mouse cytokine antibody pairs for ELISPOT applications. Please see our ELISPOT Best Protocols® page for a detailed protocol and a reference table with the list of antibody pairs that can be used in ELISPOT.
Q. I recently bought one of your Ready-Set-Go! ELISA Sets, can I use the antibody pairs to run an ELISPOT?
We do not recommend the use of our ELISA kits for the ELISPOT application. We cannot guarantee the successful use of our ELISA kits in an ELISPOT application.
Q. What are the critical parameters for successful ELISPOT assay?
It is critically important to use a high affinity binding PVDF membrane plate, rather than regular ELISA plate or nitrocellulose membrane plate. Additionally, highest affinity antibodies for capture work best in this assay. Empirical optimization of conditions such as cell density, stimulus/mitogen, and kinetics is necessary.
FlowCytomix™ Multiple Analyte Detection System
Q. Is there a particular flow cytometer required for the use of FlowCytomix™ Kits?
FlowCytomix™ microspheres are detectable in most commonly used flow cytometers. The flow cytometer needs to be equipped with one laser (488 nm or 532 nm) capable of detecting and distinguishing fluorescence emissions at 575 nm and far red (685 nm - 690 nm).
FlowCytomix™ Kits have been tested for use on:
EPICS® XLTM / XL-MCL™*
Cytomics TM FC500*
BD FACScan™
BD FACSCalibur™
BD FACSCanto™
FACSCanto™ II
BD TM LSR I
BD TM LSR II
Guava EasyCyte Plus
MACSQuant®
* For the most commonly used flow cytometers (e.g. Becton Dickinson or DakoCytomation) you can immediately start cytometer setup as below.
If you run a FlowCytomix™ assay on a FC500 Instrument from Beckman Coulter you must ensure that the Forward Scatter (FS) measurements are collected at 1-8°. To accomplish this you must insert the FS 1-8° Field Stop into place: remove the front cover to locate the FS 1-8° Field Stop. Slide the knob from the right (= default 1-19° position) to the left and push to lock in place.
On the XL Series systems the EPICS XL/XL-MCL FS Low Angle Collection Kit is recommended to accomplish this.
Q. Is special software required?
Yes, the flow cytometer raw data files should be analyzed using the FlowCytomix™ Pro Software. Download the FlowCytomix™ Pro Software for free.
Q. Does the FlowCytomix™ Pro Software also run on Apple Macintosh?
Yes, you can run FlowCytomix™ Pro Software on Apple Macintosh with Mac OS X or later.
Q. Does the Bender MedSystems FlowCytomix™ Pro Software interact with the flow cytometer operating system?
FlowCytomix™ Pro Software has been successfully operated in conjunction with all commonly used flow cytometer models. It does not interfere with the flow cytometer operating system. In addition, FlowCytomix™ Pro Software does not need to be installed on the flow cytometer working station; data files can be transferred to any other computer.
Q. Flaws in the Standard Curve - What can I do?
Flat sections in the standard curve might result in an imprecise calculation of sample values.
Small differences in MFI cause major differences in analyte concentrations. Flat parts in the low concentration range of the standard curve might be improved by removing the highest standard point, standard 1, but only if sample values are expected in the low concentration range.
If one data point is definitively an outlier (e.g fluorescent intensity of a lower standard point is higher than fluorescent intensity of a standard point with higher concentration), the data point has to be excluded. Outliers may distort the whole standard curve and the calculation of results will not be correct.
Q. What if I want to design my own multiplexing experiment?
FlowCytomix™ Simplex Kits are available for this purpose. Although FlowCytomix™ Simplex Kits are designed for the detection of one specific cytokine, they can be combined with each other provided the beads are compatible as described here: Possible Combinations
Q. When is a FlowCytomix™ Basic Kit required?
Basic Kit is required for the successful use of Simplex Kits. In order to run a FlowCytomix™ assay some reagents are needed only once, even in the case of combining several Simplex Kits. These reagents are provided in a Basic Kit.
For mouse and rat Simplex Kits the mouse/rat Basic Kit is required (cat. no. BMS8440FF).
For human Simplex Kits PE (E-selectin, ICAM-1, ICAM-3, PECAM-1, P-selectin, VCAM-1) the human Basic Kit PE (cat. no. BMS8421FF) is required. In case Simplex Kits and Simplex Kits PE are combined, the human Basic Kit is required.
For all other human Simplex Kits the human Basic Kit (cat. no. BMS8420FF) is required.
Q. Are FlowCytomix™ assays performed in plates or in tubes?
The FlowCytomix™ assay can be performed like a conventional ELISA in a 96-well filter plate or in individual tubes . When using a 96-well filter plate, a filtration manifold is required (Vacuum Manifold). The use of tubes does require centrifugation of the tubes during the washing steps.
Q. Why is a cytometer setup required before measuring FlowCytomix™ standards and samples?
The cytometer setup is required to prepare the instrument for the bead-based assays. FlowCytomix™ setup beads are included in the Basic Kits and in the Multiplex Kits to prepare the correct instrument settings. With every new experiment, before starting the acquisition of standards and samples, stay in SET UP MODE and adjust the settings using the highest standard (Standard 1).
Q. What wavelength is used for measurement?
The maximum emission of PE is at 578 nm and the emission of the beads is in the far red region (685 - 690 nm).
Q. Is it possible to measure with high "flow through" settings?
There are often three flow through settings: low, medium, & high. Increasing the "flow through" rate may result in a dispersed bead population. Therefore, it is recommended to start with the low "flow through". The rate can be increased if the bead population does not start to disperse. Once the final instrument settings are saved, do not change the "flow through" rate during measurement.
Q. Does a FlowCytomix™ Kit work on a Luminex™ instrument?
No, FlowCytomix™ Kits can only be measured on a flow cytometer.
Q. Is it correct to use the non-linear part of the standard curve during analysis?
Yes, it is possible to use the non-linear part of the standard curve for calculation of results. Dilutions of samples behave in the same way as the standard curve..
Q. How many analytes can be combined in one assay?
With the FlowCytomix™ System, it is possible to combine up to 20 analytes.
Q. Which analytes can be combined?
Up to 20 FlowCytomix™ Simplex Kits within one species can be combined. FlowCytomix™ Simplex Kits with an identical bead population cannot be combined. For possible combinations, please click here: Possible Combinations.
Q. Can FlowCytomix™ Multiplex Kits be combined with each other?
All FlowCytomix™ Human Multiplex Kits can be combined with human Th1/Th2 11plex (cat. no. BMS810FF). All FlowCytomix™ Mouse Multiplex Kits can be combined with Mouse Th1/Th2 10plex (cat. no. BMS820FF. A combination of Human Cardiovascular Panel (cat. no. BMS811FF) with Human Adhesion Panel (cat. no. BMS812FF) is possible for some analytes which do not use the same bead sets.
Q. Can FlowCytomix™ Simplex Kits be combined with a Multiplex Kit?
Yes, FlowCytomix™ Human Simplex Kits can be combined with Human Multiplex Kits provided the beads are compatible as described here: FlowCytomix Combination Table
Q. Can FlowCytomix™ components from different lots be mixed?
The FlowCytomix™ bead sets, standards and conjugates are lot-specific and must be used in combination with each other. Do not mix these components from different kit lots. FlowCytomix™ Assay Buffer, Reagent Dilution Buffer and Streptavidin-PE are not lot-specific and can therefore be exchanged between different kit lots.
Q. Is it possible to combine FlowCytomix™ Biotin Conjugates with PE-conjugates in one conjugate mixture?
Yes. However, if you are using at least one Biotin-Conjugate in your combination a further incubation step with Streptavidin-PE is necessary. This does not interfere with the PE-conjugates.