Anti-IGF1 antibody (ab9572)

Dear Kate,



Thank you for your email. As the antibodies are guaranteed to work with serum samples, what kind of pre-treatment would I need to use in order to get accurate results? What was used during testing for IGF1 ELISA?



Please find my replies to the questionnaire below in blue:







Order Details

Antibody code: ab9572 and ab83137



Problem

Choose: weak signal with serum samples but not with standard curve





Lot number GR21525-6 (ab9572) and GR9925-6 (ab83137)





Purchase order number HFE2462590-1 (ab83137) and ab9572 was sent for free as per the abpromise

or preferably Abcam order number





General Information

Antibody storage conditions (temperature/reconstitution etc)

Aliquoted ab9572 in 5ul aliquots and stored at -20C. Reconstituted ab83137 in PBS/0.1% BSA, aliquoted as 5ul aliquots and stored at -20C.



Description of the problem (high background, no signal, non-specific color development, poor standard curve etc.)



While the standard curve is good, the pre-treated serum samples are the problem. The serum is from samples that had already been tested using an IGF1 ELISA kit. This serum (from the same tube) was stored at -80C. For the current ELISA, I pre-treated the serum by first treating 30ul of serum with 120ul acid-ethanol (2.5% HCl-87.5% ethanol). This was then incubated at room temperature for 30 minutes with shaking. The samples were then centrifuged for 5 minutes at 10,000rpm and 100ul of supernatant was transferred to a fresh tube containing 200ul Tris Buffer (pH 7.6). The tube was mixed thoroughly and assayed immediately. I also assayed the same serum samples in a 1:15 dilution using PBS to check if the pre-treatment was working. Additionally, I had human serum control (from a bottle from Sigma) as an additional sample. So this was pre-treated and only diluted as well. However, the absorbance and the concentrations calculated from the standard curve for both the pre-treated samples and the diluted samples were similar and these concentrations were about 4-5-fold lower than previously obtained results for the same samples.







Type of ELISA (Direct ELISA/Indirect ELISA/Sandwich ELISA etc.)

Sandwich ELISA



Sample (Species/Cell type/Cell line etc.)

Serum samples either pre-treated or not.



Coating well (Buffer/Concentration of the coating material etc.)

ab9572 was used to coat the plates at 1:1000 dilution in PBS. The coated plates were stored at 4C overnight.



Blocking conditions (Buffer/time period, Blocking agent etc.)

5% non-fat milk in PBS solution was used to block the plates for 1 hour.



Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)

ab83137 was used as the detection antibody at 1:6000. The plates were incubated at room temperature for 90 minutes. Wash steps were done 6 times in PBS/Tween 20.



Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)

Streptavidin-HRP (Sigma) at 1:2000 dilution in PBS. Incubated for 90 minutes, wash 6x in PBS/Tween20.



Detection method (Substrate/Diluent etc.)

Citrate buffer with TMB and hydrogen peroxide.



Positive and negative controls used (please specify)

The serum samples (both pre-treated and diluted) were used as positive controls. I used two samples, one with a high concentration (˜400 ng/ml) and the other with a lower concentration (˜55 ng/ml). Also, blanks were used.







Optimization attempts (problem solving)

How many times have you tried the ELISA?

Two times with the samples; 5 times with standards and a human serum control (Sigma).





Have you run a "No Primary" control?

Yes

Do you obtain the same results every time?

The problem is not with the capture and detection antibodies not detecting IGF1 but with very low absorbance/signal for serum samples, either pre-treated or diluted.





Additional Notes:



Data:

We would appreciate if you are able to provide a copy of the data, particularly from the standard curve. This will help us to assess the results.



I have attached an Excel file with the results from the second test with samples.







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Best regards,

Kate



Kate Hayes

Scientific Support Supervisor

Abcam plc

http://www.abcam.com



Your original inquiry to Abcam:













Dear Kate,

Thank you for your reply. During testing for IGFBP3 and IGF1 antibodies, were the ELISAs tested with serum/plasma samples at all? Do you have any other customers who have used either sets of antibodies to set up a sandwich ELISA? I am having trouble with the IGF1 antibodies with pre-treated serum samples. I was wondering if you have any protocols for serum/plasma pre-treatment to use for this protocol?

Thank you,









Abcam Customer Services and Scientific Support Team

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ANSWER:

Thank you for your reply

Please note that theseantibodies have not been tested with with serum samples, and so I am sorry no pretreatments have been tested which we could provide you with information on. In theory the antibodiesshould work with serum samples. Since the antibodies are working well with their standard protein, this means that the antibodies do work in the ELISA.Therefore, I can suggestit may be that there is some interferencewith something in the serum samples.

1. Have you been able to confirm the expected amount of protein in their samples using any other method?

2. Is it possible that the samples contain a very small amount of protein?

3. Is there any azide in the samples? This could inhibit the reactionand would lead to loss ofdetection as you are observing.

4. Have you tried any other sample types?

5. Please provide details of the sample preparation.

I hope this will be helpful. Please do not hesitate to get back to me with the requested information and I hope we can resolve this for you as soon as possible.

Thank you for your reply. During testing for IGFBP3 and IGF1 antibodies, were the ELISAs tested with serum/plasma samples at all? Do you have any other customers who have used either sets of antibodies to set up a sandwich ELISA? I am having trouble with the IGF1 antibodies with pre-treated serum samples. I was wondering if you have any protocols for serum/plasma pre-treatment to use for this protocol?



Thank you,

ANSWER:

Thank you for your reply.

I would like to reassure you that ab83137 Anti-IGF1 antibody (Biotin) and ab9572 Anti-IGF1 antibody are tested and covered by our 6 month guarantee for sandwich ELISA together, and human samples. In the event that a product is not functioning in the tested applications and species 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.

As the difficulties are continuing, I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I would appreciate if you could include details of the sample pretreatment. What was the pretreatment protocol, and the reasons why this was done?

I would appreciate if you could also providethe data from the standard curve and samples which would help us to assess the results.

With regards to the protocol used for testing, both the IGF1 and IGFBP3 antibodies will have been tested using the same procedure. Once I have further information from you, I would like to investigate further this further with our source if they have any other recommendations for you from their experiences with this antibody, particularly with regards to sample type and preparation.

Thank you for your time and cooperation. We look forward to receiving the completed questionnaire.


Order Details
Antibody code:

Problem
Choose: Problem with standard curve No signal or weak signal High background


Lot number


Purchase order number
or preferably Abcam order number


General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, no signal, non-specific color development, poor standard curve etc.)


Type of ELISA (Direct ELISA/Indirect ELISA/Sandwich ELISA etc.)


Sample (Species/Cell type/Cell line etc.)


Coating well (Buffer/Concentration of the coating material etc.)


Blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method (Substrate/Diluent etc.)


Positive and negative controls used (please specify)




Optimization attempts (problem solving)
How many times have you tried the ELISA?



Have you run a "No Primary" control?


Yes No
Do you obtain the same results every time?
Yes No

What steps have you altered?


Additional Notes:



Data:
We would appreciate if you are able to provide a copy of the data, particularly from the standard curve. This will help us to assess the results.

I have been testing ab9572 together with ab83137 using a purified human IGF1 standard and some serum controls. While the antibodies work well in a sandwich ELISA when detecting the standard, I am having some trouble with the serum controls. I also tried testing two serum samples whose IGF1 levels I had previously established using an ELISA kit. However, while the original concentrations were ˜400 ng/ml and 55 ng/ml, using my ELISA, they are about 5-fold lower.

I have been using a pre-treatment protocol before analysing the serum samples/controls using the ELISA. I’ve used acid-ethanol for 30 minutes while shaking followed by neutralising it with Tris base buffer. I also tried simply diluting the same serum samples/controls using the same dilution factor as during pre-treatment and the results for the two are similar suggesting that the pre-treatment is not working.

Have you got any advice on how to best pre-treat the serum to dissociate the IGF1 from binding proteins? When the sandwich ELISA was tested, was it tested on any serum/plasma samples as well?

Thank you in advance.

Best wishes,

ANSWER:

Further to my previous response, serum samples and other biological fluids are known to show an increase in background due to the many components found in these types of samples.

Do you have the readings for the standard and test samples as well as your detailed protocol you can send to us which we could perhaps review?

Thank you for your co-operation in this matter!

Thank you for getting back to me. I look forward to your reply to my lab question.

I have been testing ab9572 together with ab83137 using a purified human IGF1 standard and some serum controls. While the antibodies work well in a sandwich ELISA when detecting the standard, I am having some trouble with the serum controls. I also tried testing two serum samples whose IGF1 levels I had previously established using an ELISA kit. However, while the original concentrations were ˜400 ng/ml and 55 ng/ml, using my ELISA, they are about 5-fold lower.

I have been using a pre-treatment protocol before analysing the serum samples/controls using the ELISA. I’ve used acid-ethanol for 30 minutes while shaking followed by neutralising it with Tris base buffer. I also tried simply diluting the same serum samples/controls using the same dilution factor as during pre-treatment and the results for the two are similar suggesting that the pre-treatment is not working.


Have you got any advice on how to best pre-treat the serum to dissociate the IGF1 from binding proteins? When the sandwich ELISA was tested, was it tested on any serum/plasma samples as well?


Best wishes,

ANSWER:

Thank you for your patience.

I try to help you as much as possible while my colleague is out of the office. I have read through all the e-mails related to your enquiry. In order to be able to identify the sources of the problem, it would be interesting to know more about the experiments:

1) Samples:

- Are you using exactly the same samples i.e. aliquotted the serum and stored in the freezer? Or the values represent the results of independent samples?

- Have you carried out a head-to head experiment with the ELISA kit and the Sandwich ELISA using the same sample?

- Are the samples from normal healthy individuals or from patients?

2) Free vs bound IGF1:

It would be interesting to know what the objectives of your studies ie. quantifying the free, or the bound or the total IGF1?

I look forward to hearing from you soon.

Hi Kate,

I've tried looking on the datasheet for ab83137 but I have not found the actual amount that is in the vial? When I bought it, it said the pack size was 50ug. I just want to confirm that this is so. I have not received it yet but obviously when it arrives I will double check what is written on it. I just want to know this now because I’m preparing a plan for the next experiment.

Thank you,

ANSWER:

Thank you for your reply.

I can confirm that one vial of ab83137 Anti-IGF1 antibody (Biotin)contains 50 ug. This information is provided at the top of the online datasheet.

This particular antibodyis provided lyophilised. The datasheet provides reconstitution instructions:

Centrifuge vial prior to opening. Reconstitute in sterile PBS containing 0.1% BSA to a concentration of 1.0 mg/ml.

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.