SciMed Technologies FAQ's
1. After adding the TMB SUBS the standards darkening too quickly, or why are the O.D. readings really high?
- This could be because when adding the enzyme to the conjugate diluent one may have added too much enzyme causing the color to darken excessively. When adding the enzyme be sure to wipe the pipette tip to remove excess enzyme coated on the side.
- The O.D. readings could also be high because the H2SO4 stop solution was not added early enough as directed.
- The plate was not properly rinsed allowing for all reagents to be cleared out.
- Always use distilled water
2. After adding the TMB SUBS why are the standards not darkening, or why are the O.D. readings low?
- This could be because when adding the enzyme to the conjugate diluent there may not have been enough enzyme added to allow for the color to change as needed. When adding the enzyme be sure to rinse pipette tip in the diluent to remove all of the enzyme coated inside pipette tip.
- The O.D. readings could be low because the H2SO4 stop solution was not added soon enough as directed.
- Always use distilled water
- Ensure your lab follows proper storage instructions ex: (store at 4șC in a fridge).
- Kit may have expired, check expiry date.
- There may have been contamination. Always use aseptic techniques when opening and removing reagents from containers. Keep the plate covered except when adding reagents, washing. Always use different pipette tips for each reagent, calibrator, control and sample (when doing duplicates never use same tip for same calibrator, control or sample). When pipetting do not allow the pipette tip to touch any of the reagents already in the well.
3. Why is there no color development?
- The enzyme was not diluted correctly into conjugate diluent.
- The kit may have expired.
- The assay may not have been followed correctly.
4. Why is there precipitate in the extracts from sample?
- When removing the upper organic layer you must make sure that the layers have separated completely or else vortex again. When pipetting the upper layer be sure to not touch the inside walls of tube and to only allow the pipette tip to touch the surface of upper layer.
5. Why is there variability with duplicates?
- One reason for this could be when rinsing the plate one well could spill to another well causing cross contamination. It is very crucial that care is taken when washing; spilling one drop or mixing wells can cause enough of a disturbance to throw readings off.
- When pipetting calibrators, controls and samples, care must be taken since only 10”l is being added. It cannot touch anywhere but the bottom of the well.
- Interruption during assay set-up. Have all reagents prepared before assay set-up commences.
- Inadequate aspiration during washing. The wells should be dumped and tapped between each washing. Tap out excess liquid in wells before adding substrate. Add substrate immediately to the wells once the excess wash buffer has been removed. If using an automated washer, ensure the machine is aspirating correctly.
6. Why is there no displacement with standard curve?
- The plate was not rinsed properly, be sure to completely rinse the plate the allotted number of times while not mixing wells.
- Standard may have deteriorated or kit has expired. If kit has not expired contact SciMed with lot number, date of expiration and OD readings.
7. The standard curve gave correct readings but the extracted samples gave low O.D. readings or no color at all.
- If this occurs with the VitaKit A ensure the final extract (upper organic layer) is diluted with hexane.
8. Does the monoclonal antibody recognize both salts, palmitate and acetate Vitamin A?
- Yes, the Monoclonal antibody recognizes both salts
9. Do the Vitakits provide reproducible quantification of vitamins at high levels? Ex: at or about 600 IU/100g of Vit D and/or 1000IU/100g of Vit A in powdered milk?
- Yes, the VitaKits can give quantitative results at high levels of Vitamins A & D.
10. Are they useful for cheese and WPC (Whole Processed Cheese)?
- We are currently developing methodology for cheese and processed cheese,.
11. Are they useful for vitamin concentrates or premixes?
- For vitamin concentrates, we are currently developing the technology.
12. Is it normal for the standard curve to be different from the insert?
- Yes, The standard curve changes proportionally because of:
- variations in a room temperature (RT),
- assay incubation time,
- volume of samples being loaded accurately, and
- washing steps.
Prepare a standard curve for each run. Do not use data from the insert or previous runs.
Summary of Typical ELISA Methodology
Potential Sources of Error
 |
 |
|||||||||||||||
|
||||||||||||||||
 |
 |
|||||||||||||||
|
|
|||||||||||||
|
||||||||||||||
|
|
|||||||||||||
|
|
|||||||||||||||
|
||||||||||||||||
|
|
|||||||||||||||
|
|
|||||||||||||
|
||||||||||||||
|
|
|||||||||||||
|
|
|||||||||||||
|
||||||||||||||
|
|
|||||||||||||