Overview

  • Product name

    Human IGFBP1 ELISA Kit
  • Detection method

    Colorimetric
  • Sample type

    Cell culture supernatant, Serum, Plasma, Cell Lysate
  • Assay type

    Sandwich (quantitative)
  • Sensitivity

    < 5 pg/ml
  • Range

    2.74 pg/ml - 2000 pg/ml
  • Recovery

    > 92 %

    Sample specific recovery
    Sample type Average % Range
    Cell culture supernatant 94.64 83% - 102%
    Serum 95.27 84% - 104%
    Plasma 92.47 82% - 102%

  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Human
  • Product overview

    Abcam’s IGFBP1 Human ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Human IGFBP1 in serum, plasma and cell culture supernatants.

    This assay employs an antibody specific for Human IGFBP1 coated on a 96-well plate. Standards and samples are pipetted into the wells and IGFBP1 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-Human IGFBP1 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 IGFBP1 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
    240X HRP-Streptavidin Concentrate 1 x 200µl
    5X Assay Diluent B 1 x 15ml
    Assay Diluent A 1 x 30ml
    Biotinylated anti-Human IGFBP1 (lyophilized) 2 vials
    IGFBP1 Microplate (12 x 8 wells) 1 unit
    Recombinant Human IGFBP1 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 1 (IGFBP1) 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 N terminal and C terminal regions are highly homologous among rat, human and bovine sequences. Contained within IGFBP1 and 2 is an integrin receptor recognition sequence (RGD) that is responsible for promoting cell migration by an IGF independent action. IGFBPs hold a central position in IGF ligand receptor interactions through influences on both the bioavailability and distribution of IGFs in the extracellular environment. IGFBPs will either inhibit or enhance the biological activities of IGF or act in an IGF independent manner.
  • Alternative names

    • AFBP
    • hIGFBP-1
    • IBP1
    • IGF-binding protein 1
    • IGF-BP25
    • Insulin-like growth factor-binding protein 1
    • Placental protein 12
    • PP12
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab100539 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

  • IGFBP1 measured in cell lysates showing quantity (pg) per mg of protein

  • IGFBP1 measured in biological fluids showing quantity (pg) per mL of tested sample

  • Representative standard curve using ab100539

  • Representative standard curve using ab100539

Protocols

References

This product has been referenced in:

See all 8 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Answer

The immunogen used to raise the antibody pair in IGFBP1 Human ELISA Kit was a recombinant human IGFBP-1, aa 26-259. (This is the full length protein minus the signal peptide).

Unfortunately, epitope mapping has not been performed so it is not known where exactly in this region the antibody pair is binding. However, based on our cross reactivity tests, this assay cross reacts ˜10% with recombinant mouse IGF-1. Therefore based on this data, it is unlikely that ab100539 will work in mouse.

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Abreviews
I tried this product on the conditioned media from the decidualized stromal cells. The product met my expectation and the protocol provided was clear and easy to use.

Abcam user community

Verified customer

Submitted Sep 03 2013

Answer

Vielen Dank für Ihren Anruf.

Leider ist es so, wie ich befürchtet habe, wir können leider keine Angabe zu diesen Kits mit HepG2 Zellen, bzw mit anderen Leberzelllinien machen. Grundsätzlich spricht aber nichts gegen die Verwendung dieses Kits mit Zellkulturlysaten.



"GENERAL TIPS FOR SAMPLE PREPARATION
Note: In case follow-up experiments are needed, it is strongly recommended to sub-aliquot all samples after preparation to minimize cytokine degradation from multiple freeze-thaw cycles.


How do I prepare conditioned media samples?
For testing conditioned medium, it is best to prepare serum-free or low serum medium as most
serum-containing media will innately contain cytokines. If testing serum-containing medium, it is
recommended to also run an uncultured media blank sample to assess the baseline responses.


1. On day 0, seed ˜1 million cells in 100 mm tissue culture plate with complete medium.*
2. On day 3, remove medium and replace medium with 6-8 ml of serum-free or low serum
containing medium (e.g. medium containing 0.2% calf serum).
3. On day 5, collect medium into 15 ml tube. Centrifuge at 2,000 rpm in centrifuge at 4ºC for 10
minutes. Save the supernatant. Transfer the supernatant into 1.5 ml Eppendorf tubes. Store
supernatant at -80ºC until experiment. Most samples can be stored this way for at least a year.


*Cell number may be lower or higher than this depending on the cell line so the optimal number will
need to be determined by each customer empirically based on researched literature and knowledge
of the particular samples.

How do I prepare cell or tissue lysate samples?
Cell or tissue lysates for use with ELISA kits can be prepared using
most conventional methods, e.g. homogenization of cell or tissue in Lysis Buffer. We supply a
compatible lysis buffer in many of our kits but other general low-salt (700 mM) lysis buffers can be
used with the following caveats:


1) Avoid using >0.1% SDS or other strongly denaturing detergents. In general, non-ionic
detergents such as Triton X-100 or NP-40 are best, although zwitterionic detergents such as
CHAPS, or mild ionic detergents such as sodium deoxycholate will work.
2) Use no more than 2% v/v total detergent
3) Avoid the use of sodium azide
4) Avoid using >10 mM reducing agents, such as dithiothreitol or mercaptoethanols


Note: In general, any buffers used for immunoprecipitations, including RIPA buffer, should work.


We strongly recommend adding some type of protease inhibitor “cocktail” to the lysis buffer prior to
homogenization. Most general biochemical supply companies stock a wide variety of these products. Since susceptibility to proteolytic cleavage and the type of proteases present in the lysate vary, we do not recommend a specific product. Instead, your choice of which combination of protease inhibitors to use should be based upon a literature search for your protein(s) of interest and/or tissue or cell type. Phosphatase inhibitors may be used but are not necessary unless the antibodies used in the kit specifically recognize phosphorylated (activated) forms of the protein.


Choices of the method for lysis and homogenization include glass-bead “smash,” douncing, freezethaw,
sonication and crushing frozen tissue with a mortar and pestle, or even a combination of these.
There is no one “best method” for all sample types, but some are better than others for some sample
types. Your choice of method should be made following a brief search of the literature to see how
samples similar to yours have been prepared in previous investigations.


After homogenization, centrifuge the lysates to remove cell/tissue debris (5 min @ 10,000 x g or 10
min @ 5,000 x g) and save the supernatant. Unless testing fresh, lysates should be frozen as soon as
possible and stored at -20°C (or -80°C, if possible). Centrifuge them again before incubating with any
immunoassay. Next, determine the protein concentration of your lysates using a total protein assay
not inhibited by detergents (such as the Bicinchoninic acid (BCA) assay) and normalize the volume of
each sample used to deliver the same amount of total protein for each assay.


Note: The Bradford assay is not recommended as it can be inhibited by the presence of detergents.


Since different cells and tissues may contain different amounts of protein, as starting point, we
suggest using 500 uL of lysis buffer per 1x106 cells or 10 mg tissue. You may have to adjust this
based upon your results. Your target for total protein concentration of the homogenate should be at
least 1,000 ug/mL, but 2,000 ug/mL or more would be better."

Ich hoffe, dies hilft Ihnen weiter. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

Benutzen Sie unsere Produkte? Schicken Sie uns einen Abreview. Verdienen Sie sich eine Belohnung!
https://www.abcam.com/abreviews

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Answer

Vielen Dank für diese Rückmeldung (wir freuen uns sehr über das Lob!).

Wie besprochen, habe ich eine kostenlose Ersatzlieferung an die ursprüngliche Lieferadresse für Sie in Auftrag gegeben. Sie hat die Referenznummer x und sollte in den nächsten Tagen bei Ihnen ankommen.

Bitte lassen Sie uns in jedem Fall wissen, wie der Ersatz funktioniert, da wir dem Problem auf den Grund gehen möchten.

Auch der Ersatz ist von unserer Garantie abgedeckt; sollte es also wieder Probleme geben, werden wir eine Lösung für Sie finden.

Ich wünsche Ihnen viel Erfolg für Ihr Projekt. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

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Answer

Vielen Dank für Ihre Anfrage und dafür, dass Sie sich die Zeit genommen haben, unseren Fragebogen auszufüllen. Diese Angaben sind für unsere Qualitätskontrollen sehr wichtig.

Es tut mir leid, dass Sie diese Probleme mit unserem IGFBP1 Human ELISA Kithatten!

Leider können wir uns nicht erklären, warum der Standard bzw. Ihre Proben mit dem letzten Kit nicht funktioniert haben. Zwar sollte der Standard nach Rekonstitution bei -20ºC oder -80ºC gelagert werden. Wenn Sie das Kit allerdings sofort verwendet haben, ist das ja nicht nötig. Und es würde auch nicht erklären, warum Ihre Proben kein Signal ergeben.

Ich weiß, dass Sie schon viel Zeit in Ihre Experimente investiert haben und es ist enttäuschend, dass Sie bisher noch kein zufriedenstellendes Ergebnis haben.Daher möchteich Ihnen anbieten, das fehlerhafte Kit zu ersetzen oder gutzuschreiben. Welche Alternative würden Sie bevorzugen?

Vielen Dank für Ihre Kooperation und Hilfsbereitschaft. Ich freue mich bald wieder von Ihnen zu hören.

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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.

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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

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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.

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Answer

Thank you for your message and cooperation, and for kindlyproviding this further information.

Reviewing the details, I appreciate the time you have spent on these experiments and would be pleased to arrange afree of charge replacementor credit note in compensation in this case.

I look forward to hearing from you with details of how you would like to proceed.

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Question

Dear Sir/Madam,

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
satisfying.


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.

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Answer

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.

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