Human IGF1 ELISA Kit (ab100545)

This kit is designed to measure free IGF1. IGF-1 is usually bound to IGFBPs in the serum/plasma, so if you would like to measure total IGF1, it is advisable to release the analytes by performing an extraction procedure such as acid-ethanol treatment (Please see below for recommended sample pretreatments).


Reagents to Pretreat Serum/Plasma Samples

Acid-ethanol extraction solution: add 2.5 ml hydrochloric acid (37% HCl)

into 10 ml deionized or distilled water, and then add ethanol to bring up to

final total volume 100 ml. Mix well.

2 M Tris buffer (pH=7.6): Dissolve 24.2 g Trizma base in 70 ml deionized

or distilled water. Adjust to pH=7.6. Bring up to final total 100 ml.


Sample Pretreatment Procedure

To release IGF-I from binding proteins, please follow the procedure outlined

below. The following procedure is only for serum and plasma sample

pretreatment. Do not pretreat cell culture supernate samples or standard.

1. Add 30 μl serum or plasma to 120 μl acid-thanol extraction solution in a

polypropylene tube.

2. Vortex and incubate for 30 min at RT with shaking.

3. Centrifuge 5 min at 10,000 rpm and transfer 100 μl supernatant into 200 μl

Tris buffer (pH=7.6) in a polypropylene tube. Mix tube thoroughly.

4. Add 300 μl Assay Diluent A. Mix well. Assay immediately.

Note: Since the samples are pretreated, the concentration read from the

standard curve must be multiplied by the dilution factor, 30.

Unfortunately the immunogen sequence for the antibodies used in the kit ab100545 is not available. Nor do we have cross reactivity data for kangaroo species. If it helps, the capture antibody used is monoclonal and the detection antibody is polyclonal.

In general IGF-1 is a fairly well conserved protein among species so cross species testing may be worth a try.

L’anticorps de capture de ce kit ne reconnait pas l’IGF-1 du rat. Pour l’anticorps de détection, la réactivé avec la protéine du rat n’a pas été testé donc malheureusement, ce kit ne peut pas être utilisé sur des échantillons de rat.

Cependant, les anticorps utilisés dans IGF1 Mouse ELISA Kit (ab100695) ont des chances de reconnaitre la protéine du rat et nous avons eu un retour positif d’un de nos clients qui a testé ce kit avec du sérum de rat.

Nous ne pouvons pas encore garantir que ce kit fonctionnera sur des échantillons de rat par contre si vous souhaitez le tester chez la rat, il me sera possible de vous offrir une remise. Après soumission d’une Abreview avec vos résultats, un avoir de la valeur du kit testé vous sera offert.

The host species for the capture antibody is mouse and the detection antibody is goat.

Vielen Dank für Ihre Antwort und dafür, dass Sie sich die Zeit genommen haben, unseren Fragebogen auszufüllen.

Es tut mir leid, dass Sie Probleme mit diesem Kit hatten. Da die Standardkurve gutgelungen ist, wird höchswahrscheinlich eine Optimierung der Proben helfen.

Um Ihr Problem zu lösen, möchte ich die folgenden Vorschläge zu Veränderung Ihres Protokols aus unserem Labor weitergeben:



In regards to the data, I agree, the standard curves looks good with strong signals strength, nice linearity, and low duplicate %CV. There looks to be some signal saturation/plateauing near the top of the curve but these saturation points can simply be eliminated. It also looks like the customer may be losing some sensitivity near the lower end of the curve as the background is slightly high, so reducing the background may help the customer detect the low levels of IGF-1 in the samples better. See attached tips for the common causes of high background and how to reduce it.

Let me ask you this – was there an acid extraction step in the customer’s protocol? I ask because about 2 months ago (near the time the customer purchased the kit) the manual was revised and this step was removed. There was nothing wrong with performing the acid extraction, it just didn’t seem to make a significant difference in most customer’s samples. However, for some customer’s samples it did, so the customer may want to consider it if not already done.

If the customer has already concentrated their samples, then other ways to increase the overall signals is to perform the optional standard/sample incubation overnight. (Keep in mind that longer incubations also may increase the background so it is even more important that the washing steps are carried out precisely when doing overnight incubations).

Others options the customer could consider to increase the sample signals are:

Increase amount of biotinylated antibody (by 1.5 fold or so – too much may increase background)
Incubate biotinylated antibody overnight (also at 4 degrees C)
Increase amount of HRP-streptavidin (by about 1.5 fold or so – too much may increase background)



Please note: it’s best to try just one of these modifications at a time. Implementing too many of these changes at once may cause high background.

The customer may also want to try initial seeding more cells to begin with and/or culturing the cells longer. They could also try different culturing treatments or media to see if that helps. If all else fails, the customer may simply have to report the IGF-1 levels in their samples are below the sensitivity limit which is 0.3 ng/ml.

Lastly, keep in mind that IGF-1 is usually bound to IGFBPs which means free IGF-1 is usually at very low levels. I wouldn’t expect the customer have the same issues with MICB or VEGF-C though.

Common Causes of High Background



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.
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.
Long incubation times in some steps (i.e. overnight sample/standard incubation) can cause high background.
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).



Bitte lassen Sie mich wissen, ob wir Ihnen helfen konnte und zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

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.

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Thank you for your enquiry.

ab100664 VEGFC Human ELISA Kit :

I can confirm this kit recognizes the mature form of VEGF-C (aa 103-227). Based on sequence similarity, the kit is also expected to detect the pro-form but this has not been specifically tested.

ab100545 IGF1 Human ELISA Kit :

This kit detects the active/free form of IGF1. However, it is not known whether the kit will also recognize the bound form (i.e. IGF1-IGFBP complex) as this has not been specifically tested or analyzed. There is an extraction step in the protocol which will helps release bound IGF-1 so if you need to detect the total IGF-1 in the samples, I would recommend including the extraction procedure. However, if you want to detect only free IGF-1 innately in the samples, you wouldn’t need to do the extraction (although I would expect the levels to be very low, likely below the sensitivity limit).

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

Thank you for contacting us. I have been in contact with the lab regarding your inquiry.They are not familiar with the Immulite 1000. However, as long as the system can perform the protocol as written, we do not see why it would not work. As this system does not use microplates, you may find it may be helpful to know the specifications of the ELISA microplate we use. T I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information. Use our products? Submit an Abreview. Earn rewards! https://www.abcam.com/abreviews