Overview

  • Product name

    Human IGFBP3 ELISA Kit
    See all IGFBP3 kits
  • Detection method

    Colorimetric
  • Sample type

    Cell culture supernatant, Serum, Plasma
  • Assay type

    Sandwich (quantitative)
  • Sensitivity

    < 80 pg/ml
  • Range

    74.07 pg/ml - 18000 pg/ml
  • Recovery

    97 %

    Sample specific recovery
    Sample type Average % Range
    Cell culture supernatant 97.46 86% - 107%
    Serum 97.46 85% - 105%
    Plasma 96.69 84% - 106%

  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Human
  • Product overview

    Abcam’s IGFBP3 Human ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Human IGFBP3 in serum, plasma and cell culture supernatants.

    This assay employs an antibody specific for Human IGFBP3 coated on a 96-well plate. Standards and samples are pipetted into the wells and IGFBP3 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-Human IGFBP3 antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of IGFBP3 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

  • Notes

    Optimization may be required with urine samples

  • Tested applications

    Suitable for: Sandwich ELISAmore details
  • Platform

    Microplate

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 1 x 96 tests
    20X Wash Buffer Concentrate 1 x 25ml
    320X HRP-Streptavidin Concentrate 1 x 200µl
    5X Assay Diluent B 1 x 15ml
    Assay Diluent A 1 x 30ml
    Biotinylated anti-Human IGFBP3 (lyophilized) 2 vials
    IGFBP3 Microplate (12 x 8 wells) 1 unit
    Recombinant Human IGFBP3 Standard (lyophilized) 2 vials
    Stop Solution 1 x 8ml
    TMB One-Step Substrate Reagent 1 x 12ml
  • Research areas

  • Relevance

    Insulin like growth factor binding protein 3 (IGFBP3) is a member of the superfamily of insulin like growth factor (IGF) binding proteins which include six high affinity IGF binding proteins (IGFBP) and at least four low affinity binding proteins referred to as IGFBP related proteins (IGFBPrP). The IGFBP members are cysteine rich proteins with conserved cysteine residues clustered in the amino terminal and the carboxy terminal regions of the molecule. The cDNA sequence encoding the mature human IGFBP3 is fused to the signal peptide of CD33. Human IGFBP3 is the major IGF binding protein in plasma where it exists in a ternary complex with IGFI or IGFII and an acid labile subunit. IGFBPs hold a central position in IGF ligand-receptor interactions through influences on both the bioavailability and distribution of IGFs in the extracellular environment. Insulin like growth factor binding protein 3 (IGFBP3) can modulate the mitogenic and metabolic effects of the insulin like growth factors (IGFs). Insulin like growth factor binding protein 3 (IGFBP3) is expressed in multiple tissues. The highest expression level is found in the non paranchymal cells of the liver. The expression levels are higher during extrauterine life and peak during puberty. Insulin like growth factors (IGFs) and IGF binding proteins (IGFBPs) play important roles in cell growth and differentiation. IGFBP3 is one of the factors in serum that is responsible for high serum induced apoptosis in PC3 cells, a prostate cancer cell line. IGFBP3 is important in controlling glucose homeostasis with increased urinary levels in type I diabetes with persistent microalbuminuria.
  • Cellular localization

    Secreted
  • Alternative names

    • Acid stable subunit of the 140 K IGF complex
    • Binding protein 29
    • Binding protein 53
    • BP 53
    • BP53
    • Growth hormone dependent binding protein
    • IBP 3
    • IBP3
    • IGF binding protein 3
    • IGFBP 3
    • Insulin Like Growth Factor Binding Protein 3
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab100541 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Sandwich ELISA Use at an assay dependent concentration.

Images

  • Representative standard curve using ab100541

Protocols

References

This product has been referenced in:

  • Nedic O  et al. Detection and identification of oxidized insulin-like growth factor-binding proteins and receptors in patients with colorectal carcinoma. Free Radic Biol Med 65:1195-200 (2013). Read more (PubMed: 24051179) »
See 1 Publication for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question


as you have an alternative way of transporting, we will leave this
arrangement to you. As we do not have an experience with such type of
payment/delivery, I must ask whether you are going to apply for import
permission from our Ministry of Health as well or we do it?
Since we had trouble with ELISA kits that were purchased, we would prefer to
replace them with:
1. rIGFBP-1 protein, 25 ug, ab50216, 325 $
2. rIGFBP-2 protein, 20 ug, ab63223, 336 $
3. BCA Protein quantification kit, ab102536, 260 $
Our purchase was 940 $ and the value of the items for replacement is 921 $.
Would this alteration be all right for you?
If we are to obtain import permission, please let me know.
With regards,
Olgica
Olgica Nedic, PhD
Head of Department for Metabolism
Institute for the Application of Nuclear Energy - INEP
University of Begrade
Banatska 31b
11080 Beograd-Zemun
Serbia
On Tue, 20 Mar 2012 14:51:26 +0100, Dragana Lagundzin wrote
> ---------- Forwarded Message -----------
> From: technical@abcam.com
> To: draganal@inep.co.rs
> Sent: Mon, 19 Mar 2012 17:12:10 +0000 (GMT)
> Subject: Reply from Abcam to your enquiry regarding ab100539,
> ab100541 [CCE3610010]
>
> Dear Sir/Madam
>
> Thank you for your message.
>
> We have a prefered courier, FedEx, who can make all these customs
> arrangments and they will invoice Abcam the customs charges which we
> will pay. You need to take no further action in this case, we are
> happy to arrange this all for you.
>
> It seems you are now unsure which items you want as a free of charge
> replacement. I can provide free of charge replacments of ab100539
> IGFBP1 Human ELISA Kit and ab100541 IGFBP3 Human ELISA Kit .
> Otherwise, I can arrange a credit note to the value of these
> products which you can use to pay for future purchases. Please
> confirm which you would prefer.
>
> I hope this will be helpful to you. If you have any further
> questions, please do not hesitate to contact me.
>
> Help us improve our service.
> Rate your experience with us today.
>
> Best regards,
> Kate
>
> Kate Hayes
> Scientific Support Supervisor
> Abcam plc
> www.abcam.com
>
> Your original inquiry to Abcam:
>
> Dear Ms Hayes,
>
> we can continue the arrangement without Dragana. We still have not
> decided what will be items for replacement. As for charges, my
> institute directly obtains import permissions for desired items and
> a company called "Spedikus" has an office at airport and it takes
> the shipment, pays customs and tax and sends us a bill with these
> expenses together with their own. If I have understood properly, you
> would like to contact and pay to "Spedikuds" directly? Should I
> provide you with some contact details (person, telephone)?
>
> With regards,
> Olgica Nedic
>
> On Sat, 17 Mar 2012 11:32:29 +0100, Dragana Lagundzin wrote
> > ---------- Forwarded Message -----------
> > From: technical@abcam.com
> > To: draganal@inep.co.rs
> > Sent: Fri, 16 Mar 2012 15:50:30 +0000 (GMT)
> > Subject: Reply from Abcam to your enquiry regarding ab100539,
> > ab100541 [CCE3605462]
> >
> > Dear Sir/Madam
> >
> > Thank you for your message.
> >
> > I can confirm we can arrange payment of the customs charges from
> > here at Abcam. There is no requirement for you to take any action.
> >
> > As your colleague will be away for 2 weeks, please let me know if
> > you would like me to arrange these kits to be sent now, or if I
> > should wait to hear from your colleague Dragana when she returns
> > from holiday.
> >
> > Thank you for your time. I look forward to hearing from you.
> >
> > Help us improve our service.
> > Rate your experience with us today.
> >
> > Best regards,
> > Kate
> >
> > Kate Hayes
> > Scientific Support Supervisor
> > Abcam plc
> > www.abcam.com
> >
> > Your original inquiry to Abcam:
> >
> > Dear Ms Hayes,
> >
> > my colleague Dragana Lagundzin will be away for 2 weeks, so I will
> > continue the correspondence.
> >
> > I confirm that we are interested for a free of charge replacement
> > for both ab100539 and ab100541 ELISA kits, together with covered
> > import cost.
> >
> > Before starting the whole procedure I have contacted our financial
> > department to enquire about the mode of payment of import cost. They
> > have communicated with a bank. For any payment from abroad we must
> > present a formal document and the suggestion was to send you a
> > proforma charging you with import cost. Is this procedure acceptable
> > for you? If yes, should the sum be in $ or E? If no, please suggest
> > an alternative.
> >
> > With regards,
> >
> > Olgica Nedic, PhD
> > Head of Department for Metabolism
> > Institute for the Application of Nuclear Energy - INEP
> > University of Begrade
> > Banatska 31b
> > 11080 Beograd-Zemun
> > Serbia
> > E-mail: olgica@inep.co.rs
> >
> > ---------- Forwarded Message -----------
> > From: "Dragana Lagundzin"
> > To: olgica@inep.co.rs
> > Sent: Tue, 13 Mar 2012 14:02:56 +0100
> > Subject: Fw: Reply from Abcam to your enquiry regarding ab100539,
> > ab100541 [CCE3588380]
> >
> > ---------- Forwarded Message -----------
> > From: technical@abcam.com
> > To: draganal@inep.co.rs
> > Sent: Mon, 12 Mar 2012 15:28:19 +0000 (GMT)
> > Subject: Reply from Abcam to your enquiry regarding ab100539,
> > ab100541 [CCE3588380]
> >
> > Dear Sir/Madam
> >
> > Thank you for your message.
> >
> > I appreciate the time you have spent on these experiments in the
> > laboratory. I am sorry to hear you are disappointed with our
> > service. I fully understand your concerns and it is disappointing
> > the results have not been successful.
> >
> > I would like to reassure you that ab100541 IGFBP3 Human ELISA Kit is
> > tested and covered by our 6 month guarantee. We are happy to offer a
> > refund, credit note or free of charge replacement when a product is
> > not working in a successfully tested applications or species within
> > the guarantee period. However, we do often find that suggesting some
> > scientifically thought out optimization tips helps to improve the
> > results. So I hope you can understand that we like to offer the best
> > technical support possible to provide a satisfactory outcome.
> >
> > As you are aware, I have been corresponding with the originator of
> > this kit. In the last email, I wanted to supply the last pieces of
> > advice and feedback they had and hoped this may be helpful to you.
> > At the end of my last email, a free of charge replacement was
> > offered, and as requested I would be pleased to arrange this for you
> > immediately. As previously discussed, as an exception in this case I
> > can arrange for any customs charges to be paid by Abcam.
> >
> > Before I process this, please let me know if you would also like the
> > free of charge replacement for ab100539 put onto this order.
> >
> > Thank you for your understanding and your continued cooperation. I
> > am sorry you have had such problems with two of our kits, and hope
> > this will provide a satisfactory outcome. I look forward to hearing
> > from you with details of how you would like to proceed.
> >
> > Help us improve our service.
> > Rate your experience with us today.
> >
> > Best regards,
> > Kate
> >
> > Kate Hayes
> > Scientific Support Supervisor
> > Abcam plc
> > www.abcam.com
> >
> > Your original inquiry to Abcam:
> >
> > Dear Mrs Hayes,
> >
> > It seems that we are going in circles with this communication. Here
> > are, once again, responses to your comments, some of them were
> > already sent before.
> >
> > 1. Yes, the function is flat, as we have reported.
> >
> > 2. Yes, some IGFBP-3 was detected, as we have reported. In your
> > manuel there are no recommendations about the dilution of samples.
> > The suggestion to dilute serum samples 20-200 times instead of 500
> > to be close to the middle of the function is not a good idea.
> > Mathematics: concentrations of IGFBP-3 in normal sera are app. 3000-
> > 6000 ng/mL, by dilution of 500 we reach 6-12 ng/mL. The first
> > standard in your kit is 18000 pg/mL or 18 ng/mL, second 6 ng/mL and
> > so on. Therefore, to reach the middle of your curve (2 or 0,67 ng/mL)
> > we could dilute samples even more, not less. By diluting serum 200
> > times we would reach concentrations 15-30 ng/mL which is at the top
> > or above the range of the supposed standard curve.
> >
> > 3. As we have written to you before, we had performed IGFBP-1 assay
> > at 4 oC, at room temperature and finally (although not recomended in
> > your manuel) at 37 oC in order to increase OD values. As OD values
> > in IGFBP-1 assay at 4 oC were much lower than at other two
> > temperatures, we did not feel we should go backwords with
> > temperature in the case of IGFBP-3, as we have already obtained very
> > low ODs at room temperature.
> >
> > 4. As we have reported before, there was only 2 uL of conjugate and
> > we used 1 uL in each of the experiments. According to your
> > recommendations, diluted conjugate is not to be stored and used next
> > day. Therefore, there is no more conjugate!
> >
> > 5. Yes, we are aware that duplicate samples are always recommended,
> > but running duplicates can only improve accuracy and not change the
> > general trend of the function.
> >
> > I believe that it is time to admit that the fungus (which you did
> > not mention at all!) that grew and metabolised in the buffer (for
> > some time, as it was quite big) most likely changed the properties
> > of the solution and, further the reactivity of antibodies and
> > conjugate that were diluted in it, resulting in significantly
> > reduced sensitivity of the assay.
> >
> > I think that it is time to arrange a free of charge replacement.
> > Please let me know whether you agree.
> >
> > With respect,
> >
> > Olgica Nedic, PhD
> > Head of Department for Metabolism
> > Institute for the Application of Nuclear Energy - INEP
> > University of Begrade
> > Banatska 31b
> > 11080 Beograd-Zemun
> > Serbia
> > E-mail: olgica@inep.co.rs
> >
> > Abcam Customer Services and Scientific Support Team
> > www.abcam.com/technical
> >
> > [CCE3588380]
> >
> > Discover more at abcam.com
> > ------- End of Forwarded Message -------
> >
> > --
> > Open WebMail Project (http://openwebmail.org)
> > ------- End of Forwarded Message -------
> >
> > --
> > INEP - Institute for Application of Nuclear Energy
> > (http://www.inep.co.yu) INEP - Institut za Primenu Nuklearne
> > Energije (http://www.inep.co.yu)
> >
> > Abcam Customer Services and Scientific Support Team
> > www.abcam.com/technical
> >
> > [CCE3605462]
> >
> > Discover more at abcam.com
> > ------- End of Forwarded Message -------
> >
> > --
> > Open WebMail Project (http://openwebmail.org)
>
> --
> INEP - Institute for Application of Nuclear Energy
> (http://www.inep.co.yu) INEP - Institut za Primenu Nuklearne
> Energije (http://www.inep.co.yu)
>
> Abcam Customer Services and Scientific Support Team
> www.abcam.com/technical
>
> [CCE3610010]
>
> Discover more at abcam.com
> ------- End of Forwarded Message -------
>
> --
> Open WebMail Project (http://openwebmail.org)
--
INEP - Institute for Application of Nuclear Energy (http://www.inep.co.yu)
INEP - Institut za Primenu Nuklearne Energije (http://www.inep.co.yu)

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Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for the following alternativeitems:


1. rIGFBP-1 protein, 25 ug, ab50216
2. rIGFBP-2 protein, 20 ug, ab63223
3. BCA Protein quantification kit, ab102536

Order number: ######

Customs authorisation will be arranged and paid by Abcam/ FedEx.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Read More

Question

here are requested information and data.
With regards,

Dear Sir/Madam

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?
Yes

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
experiment)
Column 3 - 8 different serum samples diluted 1:500, new samples, not frozen
before testing
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

Read More
Answer

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.

Read More

Answer

Thank you for your patient. I just had a response from my lab colleagues. They confirmed that all three kits were designed against the mature form of the protein (which is the full amino acid sequence minus the signalling peptide). As the epitope is located somewhere on the mature form, the detection antibodies will both recognise uncleaved and cleaved forms of the protein. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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