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here are requested information and data.
WE PERFORMED THE TEST WITH STANDARDS AND SEVERAL SAMPLES (HUMAN SERA
DILUTED1:500, AS THE EXPECTED CONCENTRATIONS OF IGFBP-3 IN SERA ARE SEVERAL THOUSAND ng/ml) IN TWO ROWS ACCORDING TO THE MANUAL (IN RESPECT TO INCUBATION TIMES, ANTIBODY AND CONJUGATE DILUTION), AT ROOM
TEMPERATURE. THE RESULTS OF THIS TEST ARE IN ATTACHMENT - FILE NAME
ELISA IGFBP-3 INEP (1);. IN THE FIRST LEFT ROW ARE STANDARDS (THE
MOST CONCENTRATED, 18 ng/ml, IN THE PLACE 2 AND ONWARD)
It sounds like the standards are in column 1 and samples are in column 2. Please confirm if this is correct If so, could you clarify which data corresponds to which standard, see attachment. Can you clarify this, see first attachment?
Column 1 - standards: 0 (position 7 in the table), 18, 6, 2, 0.667, 0.222,
0.074 and 0 (position 14 in the table) ng/ml
AND BUFFER FOR NSB (PLACES 1 AND 8, STANDARD 0).
Please confirm what is NSB?
NSB: non-specific binding obtained with standard 0
IN THE SECOND ROW ARE SERUM SAMPLES (1:500).
8 different serum samples, each diluted 500-fold, is this correct?
SINCE OD VALUES WERE LOW WE REPEATED THE TEST AT 37 oC
The excel file says the temperature 25.5ºC. What temperature was the Results 2 data ran at? Can you also clarify the identities of the standards and samples for these too?
Colum 1 - standards: 72 (position 7 in the table), 36, 18, 6, 2, 0.667, 0.222
and 0.074 ng/ml (position 14 in the table)
Column 2 - 8 different serum samples diluted 1:500 (the same as in the first
Column 3 - 8 different serum samples diluted 1:500, new samples, not frozen
The excel file 2 states the temperature at which the reading was performed -
25.5 oC (VICTOR reader, PekinElmer), whereas incubations during reaction were
carried out at 37 oC
Asked on Mar 09 2012
Thank you for taking the time to provide the further information and for your patience while we have reviewed this case with the originator. I appreciate your cooperation.
The originator has advised the following:
1. It looks like in the first experiment the standard was run in duplicate but the other standards just once. In any case, the signal strength of the standards is much lower than we would expect and there is no linear response, the curve is basically flat.
2. For the samples in the first experiment, most of them are above the background values so IGFBP-3 is being detected, just at low levels. However, sincethe recommended serum dilution range for this kit is 20-200 fold, I would suggest to decreases the dilution factor from 500-fold to increase the samples signals a little more so that when the curve improves, the samples will fall closer to the middle of it for a most accurate measurement.
3. In the second experiment, increasing the experimental temperature from room temperature to 37ºC did increase the overall signal on the whole plate (including the background). However, the curve is still flat and the samples are still low or near background levels (all the values are just higher compared to the first experiment). Please note that we do not generally advise increasing the experimental temperature.To increase the overall plate signal strength, we would advise performing the optional overnight standard/sample incubation at 4ºC.
4. We calculate there is still about a half a plate left to continuewith, is this correct? Therefore,below are some further tips on ensuring a strong signal response that could be considered. As two vials of standard (Item C) are included in the kit, you can use the fresh second lyophilized standard vial if not used already to be on the safe side.
5. Finally, please note that we also always recommend running both the standards and samples in at least duplicate for the most accurate results as well as eliminate technique error as a possible cause.
TIPS TO ENSURE A STRONG SIGNAL:
1. When preparing standards, it iscritical to briefly spin down the vial first. The powder may drop off from the cap when opening it if you do not spin down. Be sure to dissolve the powder thoroughly when reconstituting. After adding Assay Diluent to the vial, we recommend inverting the tube a few times, then flick the tube a few times, and then spin it down; repeat this procedure 3-4 times. This is a technique we find very effective for thoroughly mixing the standard without too much mechanical force.
2. Do not vortex the standard during reconstitution, as this will destabilize the protein.
3.Once your standard has been reconstituted, it should be usedimmediately or else frozen for later use.
4.Keep the standard dilutions on ice while during preparation, but the ELISA procedure should be done at room temperature.
5.Be sure to discard the working standard dilutions after use – they do not store well.
I hope this information will be helpful to you. I look forward to hearing from you with the results from the suggestions. If you are still having difficulties with the results, please do not hesitate to let me know and I will be pleased to arrange a free of charge replacement or credit note.
Answered on Mar 09 2012