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ELISA Frequently Asked Questions


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.

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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.

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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.

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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
  • 10X Coating Buffer
  • 5X ELISA/ELISPOT Diluent
  • Detection enzyme: pre-titrated Avidin-HRP
  • Substrate Solution: 1X TMB (Tetramethylbenzidine) 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.

Q. What type of plates are included in the Ready-SET-Go!® ELISA sets?

Corning Costar 96-well plates are included with Ready-SET-Go! ELISA catalog numbers ending in suffixes -22, -86 and -76. These solid 96-well plates are optional with the 10 and 20 plate kit sizes.

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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:

For ELISA pairs, eBioscience recommends using the HRP compatible substrate, Super AquaBlue (read at 405nm).  Our Ready-Set-Go! ELISA Sets include 1X TMB Solution (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.

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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
  • 10X Sterile Azide-Free Coating Buffer
  • 5X ELISA/ELISPOT Diluent
  • 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 BestProtocols 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?

A: 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.  

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