1. What is different about the FlexLISA® kit?

In a standard ELISA the capture antibody is bound directly to the surface of the well, whereas the 96 stripwell plate provided within the FlexLISA® kit is pre-coated with biotin which binds the capture antibody-streptavidin conjugate with a strong affinity. Therefore, much less capture antibody (40x less) is required compared to standard ELISA assays, saving on costs of expensive antibody.

Unlike other ELISA kits available on the market, the FlexLISA® kit also gives the flexibility of being able to use any antibody of your choice and conjugate as many capture and detection antibodies as required, giving complete flexibility.

Furthermore, the FlexLISA kit contains Expedeon’ Lightning-Link®, the world leading antibody labeling technology, for covalently conjugating your capture and detection antibody. Lightning-Link® kits enable conjugation of any antibody with a free lysine, and each Lightning-Link® kit contains 3 reactions, providing the flexibility to run multiple conjugates at the same time.

2. Are all materials required to run the FlexLISA® included in the kit?

The FlexLISA® kit contains a 96 well plate and conjugation kits to label 3 different capture antibodies and 3 different detection antibodies. Antibodies, assay buffers and enzyme detection solutions are not provided, please see the protocol for additional materials required.

3. How much label and antibody is required for this kit?

The 3x10ug Lightning-Link® labeling kits provided within the FlexLISA® kit will each conjugate 10ug of antibody which is enough to run the 96 well plate. Less than 0.1ug of capture and detection antibody is required per well and the 3 reactions provide the flexibility to run multiple conjugates at once.

4. Do I need to block the plate?

The 96 well stripwell plates are pre-coated with biotin and pre-blocked with blocking agents to prevent high background.

5. What if my sample contains biotin?

The assay can tolerate presence of biotin up to 200pg/ml in the well. If biotin levels in your sample are higher, perform a 1 hour pre-incubation with the Ab mix and then add the sample directly to the plate (no need for washing step).

6. Which substrates do you recommend?

The substrate required depends on the choice of detection label. For HRP the most common substrates are TMB, QuantaRed™ QuantaBlu™, AmpliFlu™ Red, for Alkaline Phosphatase, PNPP, 4-MUP are common place.

7. How should I store my FlexLISA kit?

The kits are shipped at ambient temperature, upon receipt make sure to store at -20°C. All the buffers provided within the kit can be stored at either 4°C or -20°C.

8. How can I avoid high background in my assay?

Please see the below table for possible causes of high background and recommended solutions.

ProblemPossible CauseRecommended Solution
High backgroundsufficient washingIncrease the number of wash steps
High backgroundInsufficient sample dilutionIncrease sample dilution
High backgroundInsufficient blockingIncrease blocking agent concentration in the assay buffer
High backgroundContaminated detection solutionRepeat assay

9. I am getting low signal in my assay, what shall I do?

Please see the below table for possible causes of signal and recommended solutions.

ProblemPossible CauseRecommended Solution
Low SignalInsufficient assay incubation timeIncrease assay incubation time to 2 - 5 hours
Low SignalInsufficient substrate solution incubation timeCheck substrate solution detection protocol (supplier)
Low SignalIncorrect plate reader settingsCheck settings used

10. What should I do about variable replicates?

Please see the below table for possible causes of variable replicates and recommended solutions.

Possible CauseRecommended Solution
Variable ReplicatesPoor pipetting techniqueCheck against best practice. We advise reverse pipetting for standard, sample and Antibody Mix loading
Variable ReplicatesContamination of samples during addition to plateCheck against best practice
Variable ReplicatesBlocked plate washerCheck all plate washer channels


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