Human TIMP1 ELISA Kit (ab100651)

Thank you for contacting us.

You are correct!, this kit comes with 12 strips of 8 wells.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for your email. I am sorry for the delay while I was in contact with lab.

The volume of HRP-Streptavidin is very small and therefore would be difficult to see with the naked eye. The HRP-streptavidin volume sent was 8ul and is very concentrated so it is especially important that the vial is spun down before opening to make sure no solution is adhering to the inside walls or cap of the vial to ensure complete recovery. Please have your researchers perform this preparation and they should be able to use the 8 ul HRP. Below are some additional tips for this component:

HRP-Streptavidin – comes as a liquid concentrate. Before withdrawing any liquid from the tube, be sure to centrifuge the vial and pipette 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.

I am also in contact with lab regarding the diluents need to be used for diluting the HRP-Streptavidin vial. I will get back to you with the information soon.

Please note, the expiration date of kit is 6 month from date of purchase including HRP-Streptavidin. Once the HRP streptavidin is reconstituted it should be used same day.

Finally, I can suggest using one kit with the provided HRP-Streptavidin, If in case it does not work then you may buy HRP- streptavidin separately. I will confirm the price if you are interested in buying this.

I hope this information will be helpful. I will contact you with more information soon.

Thank you for your inquiry and for forwarding your testing data. There are a few things I noticed after looking at the data: 1) The first thing I noticed was the background is quite high, ~0.5 OD. This high background is really reducing the dynamic range, sensitivity and linearity of the curve so this issue needs to be addressed. Please see tips below for keeping background low: Causes of High Background 1. Too much HRP-Streptavidin may cause background. Briefly centrifuge the vial of HRP-streptavidin concentrate (Item G) and pipette up and down to mix gently before using, since precipitation may form during storage. 2. Too much detection antibody may also contribute to high background. Make sure of correct dilution fold of both the biotinylated antibody (Item F) and HRP-Streptavidin. Both should be diluted with 1x Assay Diluent B. 3. Long incubation times in some steps (i.e. overnight incubation) can cause high background. 4. And finally, the wash steps may introduce background: if the plate is insufficiently washed, or if the wash buffer itself was contaminated. Wash buffer must be removed completely from the wells after each wash. We recommend using an automated plate washer or multi-channel pipettor. Squirt bottles are not advised. If the high background persists after checking all these steps, try reducing the amount of HRP-streptavidin (by increasing the dilution by 1.5 or 2-fold). 2) The second and main issue I see is the non-linear pattern of the standards, specifically from the 18,000pg/ml to the 2,000pg/ml. It would be quite difficult to obtain lower OD response from the 18,000pg/ml and 6,000pg/ml compared to the 2,000pg/ml as the standards are serial diluted. Therefore I don’t think the issue is a standard preparation issue, per se, as there is a noticeable linear pattern from 2,000pg/ml to at least the 666.7pg/ml standard. It seems to be a problem with the two most concentrated points, 6,000 & 18,000 pg/ml. If you omit those points (see lower graph attached), the standard curve is actually okay (r^2 = 0.986). Because the background is so high, it is likely that the highest two standards are reading falsely low (due to precipitate on the bottom of the wells) and that the real values are actually very high and saturated.  Was there any precipitate in the bottom of any of the wells?  If so, signal saturation is probably the issue and can occur if there is too much target protein in the well, causing the enzyme reaction to go too fast, and the color product crashes out of solution.  The most common causes of signal saturation are: 1. Waiting too long before adding stop solution (makes readings artificially high) – make sure to not let the color reaction (TMB incubation) go longer than 30 mins. 2. Doing the color development reaction at high temperature (such as 37 degrees C; also makes readings artificially high) 3. Exposing the plate to light during color development reaction step – light can speed up the reaction and cause falsely high signals. 4. Long incubations – if you incubated overnight, try decreasing to the optional shorter incubation time. 5. Too much HRP-streptavidin – the HRP-streptavidin vial comes highly concentrated so even a small change in loading volume can drastically affect the results ODs. Remember to briefly centrifuge the vial of HRP-streptavidin concentrate (Item G) and pipette up and down to mix gently before using, since precipitation may form during storage. 6. Too much detection antibody - make sure of correct dilution fold of both the biotinylated antibody (Item F) and HRP-Streptavidin. Both should be diluted with 1x Assay Diluent B. 7. Insufficient washings (or contaminated wash buffer) - wash buffer must be removed completely from the wells after each wash. An automated washer or multi-channel pippetor is recommended. Use of a squirt bottle is not advised Because signal saturation and high background are both issues here, I would advise to pay close attention to components that are factors in both, specifically excess HRP-streptavidin and too long TMB incubations. Lastly, you are experiencing some minor high duplicate CV issues which most likely will be corrected once the main issues are addressed but I’ve included some tips for keeping CV low: Causes of High CV 1. Pipette performance – be sure pipettes are calibrated and functioning properly 2. Bubbles in wells – make sure that the plates are not rocked too vigorously during incubations and that there are no bubbles present during reading. 3. Incomplete washing – ensure complete removal of wash buffer after each wash. Also, do not use a squirt bottle to wash the wells – a pipette or a plate washer is recommended. 4. Incomplete reagent mixing (particularly the biotinylated antibody). I hope this information helps. Let me know if the tips do not improve the standard curve readings.

Thank you for contacting us. This kit ab100651 will detect free TIMP-1, both pro and active form (thus, total) however it is not known whether it will also detect the bound form (i.e. TIMP-MMP complexes).   I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.