Bender MedSystems ELISA FAQs

General FAQS regarding Bender MedSystems ELISA Kits:

Which samples are suitable for the Bender MedSystems ELISAs?
Most ELISAs are suitable for human serum, plasma, cell culture supernatants and other body fluids. Detailed information is given on the datasheet and in the Bender MedSystems catalogue.

Do the standards of the Bender MedSystems ELISAs correlate with international standards?
The following standards correlate 1:1 with international reference standards:
human IFN-gamma Int. Referenz NIBSC 82/587 50pg = 1IU
human IL-2 Int. Referenz NIBSC 86/504 76pg = 1IU
human IL-5 Int. Referenz NIBSC 90/586 100pg = 1IU
human IL-6 Int. Referenz NIBSC 89/548 10pg = 1IU
human IL-10 Int. Referenz NIBSC 93/722 200pg = 1IU
human IL-12 Int. Referenz NIBSC 95/544 100pg = 1IU
human IL-13 Int. Referenz NIBSC 94/622 1ng = 1IU
human TNF-beta Int. Referenz NIBSC 87/640 6.7pg = 1IU
murine IL-2 Int. Referenz NIBSC 93/566 10pg = 1IU
murine IL-4 Int. Referenz NIBSC 91/656 100pg = 1IU
murine IL-6 Int. Referenz NIBSC 93/730 10pg = 1IU
murine IL-10 Int. Referenz NIBSC 93/672 1pg = 1IU

Do I have to run the ELISA standards and samples in duplicates?
No, but it is highly recommended to make double determinations of each sample and standard point.

Is it possible to use the reagents of other ELISA kits/lots?
Do not substitute components from other ELISA kits/lots.

Can I always use the same manual for different ELISA kit lot numbers?
Always use the manual included in the ELISA kit.

Is it necessary to wash the ELISA plates before usage?
This washing step is not necessary, but recommended as precision may be lower in case of omission.

Is shorter/longer incubation time possible?
Incubation times should be followed exactly to ensure optimal ELISA test performance.

Can I omit the dilution of my ELISA samples with assay buffer/sample diluent when performing the assay?
We recommend following the dilution-instruction provided in the manual. We can not guarantee optimal performance of the assay if internal assay dilutions as instructed were omitted.

What is to do if the reagent volume is not enough?
In case of small reagent volumes, please spin down vial before usage to make sure to collect all the contents.

Is it possible to store the reagents other than indicated?
Storage of the kit components other than indicated is not recommended in order to assure proper performance of the test.

Do you have information regarding the normal values detectable with your ELISA Kit?
Find "normal levels" in the manual of the respective kit under point "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 performs its own studies to establish its own normal range for patient populations.

ELISA Troubleshooting:

What are the reasons for high background?

  • Improper Washing:
    Insufficient washing, omission of one of the wash steps can lead to high background. Check volume of ELISA wash buffer and make sure all recommended washing steps are performed. Make sure the ELISA wash buffer out of the kit is used and diluted correctly.
    Leave the ELISA wash buffer in the wells for a few seconds.
  • Contaminated Substrate:
    Make sure there is no contamination of the substrate before usage with metal ions or oxidizing reagents. The substrate solution should not develop colour by itself.
  • Contaminated Diluent:
    Most of the ELISA diluents are based on a protein matrix. Upon first opening contamination of the ELISA diluent may occur. Use only sterilized pipettes to remove ELISA diluent needed from the bottle.
  • Substrate exposed to light:
    Exposure to light may result in a blue colour of the substrate. Keep solutions in the dark (vial) until ready to dispense into the plate.
  • Wrong Conjugate dilution:
    Make sure the conjugate is diluted according to the guidelines in the manual. Too high concentration of the conjugate might result in elevated background.
  • Wrong Incubation Times/Temperatures:
    Exactly follow the ELISA test protocol regarding incubation times and temperatures.
  • ELISA plate stored improperly:
    In case of partial usage of the plate, make sure that the microwell strips are stored properly

What are the reasons for high intra assay CVs?

  • Coating removed by scratching during washing/pipetting:
    Take care not to scratch the inner surface of the microwells when washing the plate or pipetting standards and samples.
  • Cross contamination from well to well by peeling off plate cover:
    Carefully peel off the plate cover after incubation to avoid cross contamination from one well to another.
  • Inhomogeneous samples:
    Make sure the samples you use for the test are homogeneous. Mix before addition to the ELISA plate.
  • Edge effect:
    Seal the ELISA plate during incubation and incubate on a rotator set at 100 rpm if possible.
  • Pipettes used are not properly calibrated:
    Make sure the pipettes you use are properly calibrated.
  • Improper Washing:
    Insufficient washing or omission of one of the wash steps can lead to high CV values.
    Check the volume of washing buffer and make sure all recommended washing steps are carried out. Make sure the ELISA wash buffer included in the kit is used and diluted correctly.
    Leave the ELISA wash buffer in the wells for a few seconds.
What are the reasons for incorrect standard curves?

  • Wrong reconstitution volume of lyophilized standard:
    Make sure the lyophilized powder is reconstituted in the correct volume of the correct reconstitution media. Reconstitution volumes are given in manual and/or on vial label.
  • Reconstitution time too short:
    Make sure the reconstitution process is given enough time to allow complete solubilisation of the lyophilized powder.
  • Wrong dilution of concentrated stock standard:
    Make sure the concentrated stock standard provided is diluted properly according the manual.
  • Wrong storage of stock material/pre-dilutions:
    Storage conditions of stock standard solution as well as pre-dilutions are given in the manual. Exactly follow these guidelines.
  • Contamination of standard:
    Contamination of the standard should be avoided by usage of sterile pipettes and proper storage.
  • Improper mixing at standard dilution steps:
    Preparing serial dilutions of the concentrated standard make sure there is proper mixing at each step of dilution.
  • Wrong measurement wave length:
    Make sure the photometer used is adjusted to the correct wave length. Please check for the reference wave length.
  • Standard from a different lot used:
    Use only standard of the kit to perform assay. Do not substitute the standard from another kit.

What are the reasons for low ELISA OD values?

  • Incubation time with the TMB substrate is too short:
    The time indicated in the test protocol may vary due to temperature or other assay conditions (temperature, kit age, humidity).

    Observe the colour development on the plate and use your own judgement to stop the substrate reaction. We recommend adding the ELISA stop solution when the highest standard has developed a dark blue colour.
    Alternatively you may monitor the colour development with the ELISA reader at 620 nm after adding the TMB substrate solution. The substrate reaction should be stopped (with ELISA stop solution) as soon as an OD of 0.9 - 0.65 is reached.
  • Wrong reconstitution volume of lyophilized ELISA standard:
    Make sure the lyophilized powder is reconstituted in the correct volume of the correct reconstitution media. Reconstitution volumes are given in manual and/or on vial label.
  • Reconstitution time too short:
    Make sure the reconstitution process is given enough time to allow complete solubilisation of the lyophilized powder.
  • Wrong dilution of concentrated stock standard:
    Make sure the concentrated stock standard provided is diluted properly according the manual.
  • Shaken:
    Not shaken assays will show reduced OD values.

What are the reasons for no color development:

  • Reagents not at RT at test begin:
    Bring all reagents to room temperature before performing the test.
  • Incorrect storage of individual ELISA reagents:
    Storage conditions of all ELISA kit components are given in the manual. Exactly follow these guidelines.
  • Contamination of ELISA HRP-Conjugate:
    Make sure there is no contamination of the ELISA substrate before usage.
  • Wrong dilution of ELISA HRP-Conjugate:
    In case the concentrated conjugate is diluted too high, color development will be weak. Follow the guide lines in the test manual.
  • Omission of any incubation step:
    Make sure each step of the ELISA test protocol is performed. Addition of the color giving reagents can help you follow your test performance.
  • Microwells dried out after washing or during storage:
    After washing, tap ELISA microwell strips on absorbent pad or paper towel to remove excess ELISA wash buffer. Use the microwell strips immediately after washing or place upside down on a wet absorbent paper for not longer than 15 minutes. Do not allow wells to dry.
  • Coating removed by scratching during washing/pipetting:
    Take care not to scratch the inner surface of the microwells when washing the plate or pipetting standards and samples.
  • Samples not suitable for the ELISA test:
    A list of samples suitable for the ELISA test is given in the manual. Any other sample may not be suitable for the ELISA test or assay conditions need to be adapted for that sample (e.g.other dilution).

What are the reasons for ELISA samples or standard in the overflow?

  • Wrong dilution of sample/standard:
    Exactly follow the test protocol for dilution of standard and samples.
  • Wrong dilution of conjugate:
    Exactly follow the test protocol for dilution of conjugate. Too high concentration of the conjugate may result in overflow of the OD measured.
  • Wrong diluent used:
    Use only diluent delivered with the kit to dilute samples.
  • Wrong measurement wave length:
    Make sure the photometer used is adjusted to the correct wave length. Please check for the reference wave length.
  • Development time of TMB substrate too long:
    The time indicated in the test protocol may vary due to temperature or other assay conditions (temperature, kit age, humidity).
    Observe the colour development on the plate and use your own judgement to stop the substrate reaction. We recommend adding the stop solution when the highest standard has developed a dark blue colour.
    Alternatively you may monitor the colour development with the ELISA reader at 620 nm after adding the TMB substrate solution. The substrate reaction should be stopped (with stop solution) as soon as an OD of 0.6 - 0.65 is reached.
  • Sample not suitable for the test:
    A list of samples suitable for the test is given in the manual. Any other sample may not be suitable for the test or assay conditions need to be adapted for that sample (e.g.higher dilution)
  • Contamination of sample/standard:
    Contamination of the sample or standard might result in unreadable results.
  • Wrong incubation time/temperature:
    Please follow exactly the test protocol regarding incubation time and temperature.
  • Plate covers were not applied during incubation:
    Application of plate covers during incubation prevents evaporation of liquid as well as contamination during incubation. Plate covers are supplied with each kit.

Tips and Tricks for Bender MedSystems Module Sets

  • Storage:
    The components of the Bender MedSystems Module Set must be stored at -20°C.
    After the first use aliquote the components. Use each aliquot once and discard the remaining material.
  • Plate:
    Use Maxisorp 96well plates (e.g.: Nunc Maxisorp plates)
  • BSA:
    BSA is a component of the assay buffer. BSA from different providers or different lots may lead to a significant decrease of the assay performance (high blanks, peculiar standard curves, low ODs)
  • TMB substrate solution:
    TMB from different providers may influence the assay performance (lower ODs).
  • Coating, blocking & fixation of plates:
    You coate the plate in PBS (without BSA) and you block with Assay Buffer (containing BSA). Fixation of coated plates before usage often leads to a decrease of signal.
  • Working procedure:
    Follow exactly the working procedure provided in the manual.
    Only use freshly prepared dilutions. Do not store any leftover diluted solutions.
    Mind the recommended incubation times and temperatures.

We cannot guarantee optimal performance if components others than those provided in the kit are used or if the assay is modified in any way not following the guidelines of the manual.

For additional troubleshooting please refer to the conventional Bender MedSystems ELISA information.