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We have purchased IGFBP-1 Human ELISA kit (ab100539), together with IGFBP-3
Human ELISA kit, from Abcam last month. Items were delivered to us during the last week in December.
We have started to work with IGFBP-1 ELISA kit and encountered serious
problems. We wanted to analyse serum and amniotic fluid samples and we have
prepared different dilutions. We followed Instructions for Use completely for
our first experiment and surprisingly got no colour development in the last
step when TMB was added. The first incubation was performed at 4 oC overnight.
We were slightly suspicious about a high (recommended) dilution of
HRP-Sreptavidin (1/12000) as, according to this dilution, the amount of the
supplied HRP-Sreptavidin was sufficient for 10 plates, not only one.
Therefore, in our next experiment we have made a dilution of HRP-Streptavidin
1/1200. We have incubated the reaction mixture at room temperature for 2.5 h,
as recommended as an alternative and tested whole serum samples (no dilution
was made). Still, no colour and no results.
In the third trial, we incubated the reaction mixture at 37 °C and increased the
period of each incubation step by 50 %. We have tested several standards and
samples including the standard stock solution (50 ng/mL), and used a HRP-Streptavidin
solution diluted 1/1200. This time some colour developed, but the results were not
Here are some results (A at 450 nm):
1.71 for 50 ng/ml standard,
0.20 for 2ng/ml standard,
0.13 for 222.2 pg/ml standard,
0.15 for 2.74 pg/ml standard,
0.15 for amniotic fluid (1/50000 diluted),
0.13 for non-diluted serum sample,
0.14 for serum sample 1/10 diluted and
0.16 for a dilution buffer as a sample(to test non-specific interaction).
According to these results, it seems that the test does not differentiate between
IGFBP-1 concentrations lower than 2 ng/mL, which is above the physiological range of IGFBP-1 in human serum. These
results suggest that the test has low sensitivity and can, possibly, work onlywith samples having high concentrations of IGFBP-1.
In these testings we have used half of the ELISA kit and we are asking for
advise how to make use of the rest of the test for serum samples.
Asked on Jan 17 2012
Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this kit.
The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality. I would like to reasure you that the kit is covered by our 6 month guarantee should it not be working.In the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.
I understand your concerns, and as you have tried a lot of omtimization already, I would be pleased to provide a free of charge replacment or credit note if you have purchased in the last 6 months.
However, in addition to this I have reviewed this case with the originator of the kit. They have kindly offered some suggestions basedon their experience to help optimise the results. I can suggest you may like to consider these first:
Based on theexplanation and data values, it may be the cause of the overall low OD is likely due to a problem with a detection component(s). While it would be difficult to eliminate the standard protein as a possible culprit, the fact that there was some OD response from the standard but not with the samples (all which are at background levels), tends tosuggestthe issue is with a detection component, most likely the main signal enhancing reagents, HRP-streptavidin and/or detection antibody.
Based on past experience,incorrectis the most common cause of low overall signal as this can cause components to degrade and lose their activity.However, if the kit has been stored as recommended, then otherpossiblesources of the problemcould be elminated. For example, testing the TMB and HRP-streptavidin activity.
Test for TMB and HRP-streptavidin activity:
Prepare a 100-fold dilution of HRP-Streptavidin: add 2ul HRP-streptavidin into a tube with 198 ul 1x Assay Dilent B. Mix through.
Prepare a final 100,000-fold diluted HRP-Streptavidin: pipette2 ul of prepared 100-fold diluted solution into a tube with 2 ml 1x Assay Diluent B. Mix through.
Pipette100 ul 100,000-fold diluted HRP-Streptavidin solution into a 96 well plate(your own plate or our supplied plate) and then add 100 ul TMB into the well (show blue color).
Read the OD value at 405 nm immediately. If a strong blue color appears, the HRP-streptavidin and TMB can be eliminated as the cause of the issue.
Make sure each reagent tube is spun down and/or mixed well before reconstituting or diluting.
If a strong blue color does not develop, please do contact me again and let me know.
If color does develop and the you still have thesecond fresh/unused detection antibody vial to use, I would advise to consider the following tips as you continue with the last half of the plate. (If this works, it is possible the first detection antibody vial got deactivated somehow).
Detection Antibody – once reconstituted, this must be used within a couple of days. We provide 2 tubes, enough for half a plate each. Be sure to centrifuge before reconstitution.
HRP-Streptavidin – comes as a liquid concentrate. Before withdrawing any liquid from the tube, be sure to centrifuge the vial and pipet up and down to mix thoroughly, as precipitation may form in storage. Once diluted, the HRP-strep must be used up that day. Do not store. Also, do not prepare the HRP-streptavidin with Assay Diluent A as it contains sodium azide which inhibits HRP activity.
TMB Solution - This is stable for 6 months at 4 degrees C, but it is light-sensitive.
Pre-coated ELISA plate - once the microplate wells have been opened, they must be stored at 2-8oC and used within one month. Return unused wells to the pouch containing desiccant pack, and reseal along the entire edge.
We would also suggest to decrease the sample dilution factors to try and increase the sample signals.The recommended serum dilution for this kit is 5-500 fold.
I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with details of how you woudl like to proceed.
Answered on Jan 17 2012