| 1. What is Sandwich ELISA assay? |
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It is an assay to determine the concentration of a specific protein in unknown samples.
The sandwich ELISA requires a "coating/capture antibody" which is coated on the well and a "detection antibody" that will later bind to the capture antibody-bound protein.
The assay is then quantified by measuring the amount of the developed enzyme of secondary antibody and its colorimetric substrate.
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| 2. What factors do influence the performance of ELISA? |
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The binding ability of the capture and detection antibodies to the protein.
The concentration of capture and detection antibodies.
The state of the ELISA reagents.
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| 3. What is the workflow of Sandwich ELISA? |
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· Step 1: The capture antibody of known concentration is coated on the plate.
· Step 2: The unknown sample is added to the plate, incubated and then washed.
· Step 3: The detection antibody is added, incubated and then washed.
· Step 4: Enzyme labeled secondary antibody is added, incubated and washed.
· Step 5: The substrate is added and a result is read in a spectrophotometer.
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| 4. What is competitive ELISA? |
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If two monoclonal antibodies that each bind to different epitopes of the same protein is hard to find, than competitive ELISA is a way to know the concentration of the protein in unknown sample. The capture antibody is coated on the plate and the unknown sample is added and incubated. Then enzyme-labeled immunogen is added. It will bind to any binding site of the capture antibody that is not occupied by the already bound protein. After the substrate is added you can calculate the concentration of protein based on the color. The less color, the more protein.
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| 5. What kinds of specimen samples are used with KOMA Cytokine ELISA Kit? |
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KOMA Cytokine ELISA Kits are suitable for serum, plasma, cell culture supernatants and other body fluids from Human, Mouse or rat samples.
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| 6. What is the difference between KOMA Complete kit and KOMA Core kit? |
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Whereas the Core kit contains the fundamental items for ELISA (capture and detection antibody, standard, substrate), the Complete kit contains all components for ELISA such as antibody coated plate, detection antibody, color development enzyme, assay diluting solution, stop solution, PBS powder and Tween-20.
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| 7. What is the difference between pink-ONE Cytokine ELISA kit and other conventional Cytokine ELISA? |
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The only difference is the substrate. In the pink-ONE Cytokine ELISA kit, the TMB substrate is prestained, so the pink-colored TMB makes convenient to track for reagent.
This way you can check whether you added the substrate or not. Also, you don’t need to add the substrate buffer after applying TMB.
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| 8. What are the reasons for high background? |
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 Washing was not enough.
Increase washing volumes and times.
Make sure that all washing steps are performed.
Make sure that the enough volume of wash buffer is remained in the bottle.
Leave the wash buffer in the wells for a few seconds.
Incubation time was too long.
Reduce incubation times.
Color development enzyme (Avidin-HRP) was too concentrated.
Make sure the Color development enzyme is diluted accordingly.
Kit materials or reagents ere contaminated or expired.
Make sure there is no contamination of the reagents.
Make high quality & fresh reagents.
Use sterilized pipettes.
Blocking was insufficient.
Change the blocking agent.
Increase blocking times.
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| 9. What are the reasons for No signals? |
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The ELISA step was not performed correctly.
Repeat assay.
Make sure each step of the ELISA test protocol is performed.
Color development enzyme (Avidin-HRP) was contaminated.
Make sure there is no contamination of the reagents.
Use fresh reagents.
Kit reagents were contaminated.
Make fresh buffers.
Make sure the standard is fine.
Coating/Capture antibody was not bound to the plate properly.
Make sure the plate is for ELISA application.
Take care when washing or pipetting not to remove the antibody.
Make sure the concentration of buffer is correct.
Check the antibody quality.
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| 10. What are the reasons for the incorrect standard curves? |
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The dilution of the standard was wrong.
Make sure calculations of reconstitution and dilution of the standard is correct.
The standard was contaminated.
Make sure there is no contamination of the standard.
Use sterilized pipettes.
The wavelength measurement was wrong.
Make sure the wavelength was set up correctly.
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| 11. What are the reasons for low OD values? |
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Incubation time of TMB was not sufficient.
The incubation time may vary according to temperature, humidity or other conditions. Try to find the optimal conditions experimentally.
The dilution of the standard was wrong.
Make sure calculations of reconstitution and dilution of the standard is correct.
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